Induction of AmpC-Mediated ß-Lactam Resistance Requires a Single Lytic Transglycosylase in Agrobacterium tumefaciens.

The remarkable ability of Agrobacterium tumefaciens to transfer DNA to plant cells has allowed the generation of important transgenic crops. One challenge of A. tumefaciens-mediated transformation is eliminating the bacteria after plant transformation to prevent detrimental effects to plants and the release of engineered bacteria to the environment. Here, we use a reverse-genetics approach to identify genes involved in ampicillin resistance, with the goal of utilizing these antibiotic-sensitive strains for plant transformations. We show that treating A. tumefaciens C58 with ampicillin led to increased ß-lactamase production, a response dependent on the broad-spectrum ß-lactamase AmpC and its transcription factor, AmpR. Loss of the putative ampD orthologue atu2113 led to constitutive production of AmpC-dependent ß-lactamase activity and ampicillin resistance. Finally, one cell wall remodeling enzyme, MltB3, was necessary for the AmpC-dependent ß-lactamase activity, and its loss elicited ampicillin and carbenicillin sensitivity in the A. tumefaciens C58 and GV3101 strains. Furthermore, GV3101 ΔmltB3 transforms plants with efficiency comparable to that of the wild type but can be cleared with sublethal concentrations of ampicillin. The functional characterization of the genes involved in the inducible ampicillin resistance pathway of A. tumefaciens constitutes a major step forward in efforts to reduce the intrinsic antibiotic resistance of this bacterium. IMPORTANCE Agrobacterium tumefaciens, a significant biotechnological tool for production of transgenic plant lines, is highly resistant to a wide variety of antibiotics, posing challenges for various applications. One challenge is the efficient elimination of A. tumefaciens from transformed plant tissue without using levels of antibiotics that are toxic to the plants. Here, we present the functional characterization of genes involved in ß-lactam resistance in A. tumefaciens. Knowledge about proteins that promote or inhibit ß-lactam resistance will enable the development of strains to improve the efficiency of Agrobacterium-mediated plant genetic transformations. Effective removal of Agrobacterium from transformed plant tissue has the potential to maximize crop yield and food production, improving the outlook for global food security.

Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3x Larix gmelinii 9.

To date, there are few reports of the successful genetic transformation of larch and other conifers, mainly because it is difficult to transform and integrate exogenous genes. In this study, hybrid larch Larix kaempferi 3x Larix gmelinii 9 cones were collected on June 27, July 1, July 4, July 7 and July 16, 2017. Embryogenic callus induction was studied using a combination of different plant growth regulators and concentrations. The results showed that July 1 was the best stage; the highest induction rate was 10.83%, which cultured in BM medium (Button medium, which formula was listed in S1 Table) with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L KT(kinetin). When cultured on a proliferation medium for 12 days, proliferation was the fastest, reaching 323.08%, which could also maintain the freshness and vitality. The suitable pre-culture medium for somatic embryogenesis was 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose. The combination of 45 mg/L ABA (abscisic acid) and 75 g/L PEG4000 (Polyethyene glycol 4000) could promote the number of somatic embryos, and reached the maximum, 210 140 per 1 g FW. The genetic transformation was carried out by the Agrobacterium-mediated transformation method with embryogenic callus cultured for 12 days. The results showed the optimal OD600 of the infection solution(suspension of A. tumefaciens) was 0.5, co-culture time was 2 days, and screening concentration of Hyg (hygromycin B) was 4 mg/L. In this study, the transformation rate of resistance callus was 32.1%. It provides a reference for low genetic transformation efficiency of larch at present. This study could be beneficial for the innovation and breeding of larch by genetic engineering and provides a certain basis for rapid propagation of excellent larch germplasm resources and genetic engineering breeding of larch and other conifers.

Motility control through an anti-activation mechanism in Agrobacterium tumefaciens.

Many bacteria can migrate from a free-living, planktonic state to an attached, biofilm existence. One factor regulating this transition in the facultative plant pathogen Agrobacterium tumefaciens is the ExoR-ChvG-ChvI system. Periplasmic ExoR regulates the activity of the ChvG-ChvI two-component system in response to environmental stress, most notably low pH. ChvI impacts hundreds of genes, including those required for type VI secretion, virulence, biofilm formation, and flagellar motility. Previous studies revealed that activated ChvG-ChvI represses expression of most of class II and class III flagellar biogenesis genes, but not the master motility regulator genes visN, visR, and rem. In this study, we characterized the integration of the ExoR-ChvG-ChvI and VisNR-Rem pathways. We isolated motile suppressors of the non-motile ΔexoR mutant and thereby identified the previously unannotated mirA gene encoding a 76 amino acid protein. We report that the MirA protein interacts directly with the Rem DNA-binding domain, sequestering Rem and preventing motility gene activation. The ChvG-ChvI pathway activates mirA expression and elevated mirA is sufficient to block motility. This study reveals how the ExoR-ChvG-ChvI pathway prevents flagellar motility in A. tumefaciens. MirA is also conserved among other members of the Rhizobiales suggesting similar mechanisms of motility regulation.

Agrobacterium-Mediated Transient Transformation of Marchantia Liverworts.

Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying ß-glucuronidase (GUS) genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization.

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.

Human Indoleamine 2,3-dioxygenase 1 (IDO1) Expressed in Plant Cells Induces Kynurenine Production.

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.

Development of a new Agrobacterium-mediated transformation system based on a dual auxotrophic approach in the filamentous fungus Aspergillus oryzae.

Genetic engineering of the filamentous fungus Aspergillus oryzae still requires more suitable selection markers for fungal transformation. Our previous work has shown that Agrobacterium tumefaciens-mediated transformation (ATMT) based on the uridine/uracil auxotrophic mechanism with pyrG as the selection marker is very efficient for gene transfer in A. oryzae. In the present study, we delete the hisB gene, which is essential for histidine biosynthesis, in A. oryzae via homologous recombination and demonstrate that hisB is a reliable selection marker for genetic transformation of this fungus. Under optimal conditions, the ATMT efficiency of the histidine auxotrophic A. oryzae reached 515 transformants per 106 spores. Especially, we have succeeded in constructing a new ATMT system based on dual auxotrophic A. oryzae mutants with two different selection markers including hisB and pyrG. This dual auxotrophic ATMT system displayed a transformation efficiency of 232 transformants per 106 spores for the hisB marker and 318 transformants per 106 spores for the pyrG marker. By using these selectable markers, the co-expression of the DsRed and GFP fluorescent reporter genes was implemented in a single fungal strain. Furthermore, we could perform both the deletion and complementation of the laeA regulatory gene in the same strain of A. oryzae to examine its function. Conclusively, the ATMT system constructed in our work represents a promising genetic tool for studies on recombinant expression and gene function in the industrially important fungus A. oryzae.

Type IV secretion systems: Advances in structure, function, and activation.

Bacterial type IV secretion systems (T4SSs) are a functionally diverse translocation superfamily. They consist mainly of two large subfamilies: (i) conjugation systems that mediate interbacterial DNA transfer and (ii) effector translocators that deliver effector macromolecules into prokaryotic or eukaryotic cells. A few other T4SSs export DNA or proteins to the milieu, or import exogenous DNA. The T4SSs are defined by 6 or 12 conserved "core" subunits that respectively elaborate "minimized" systems in Gram-positive or -negative bacteria. However, many "expanded" T4SSs are built from "core" subunits plus numerous others that are system-specific, which presumptively broadens functional capabilities. Recently, there has been exciting progress in defining T4SS assembly pathways and architectures using a combination of fluorescence and cryoelectron microscopy. This review will highlight advances in our knowledge of structure-function relationships for model Gram-negative bacterial T4SSs, including "minimized" systems resembling the Agrobacterium tumefaciens VirB/VirD4 T4SS and "expanded" systems represented by the Helicobacter pylori Cag, Legionella pneumophila Dot/Icm, and F plasmid-encoded Tra T4SSs. Detailed studies of these model systems are generating new insights, some at atomic resolution, to long-standing questions concerning mechanisms of substrate recruitment, T4SS channel architecture, conjugative pilus assembly, and machine adaptations contributing to T4SS functional versatility.

Exposure of Agrobacterium tumefaciens to agroinfiltration medium demonstrates cellular remodelling and may promote enhanced adaptability for molecular pharming.

Agroinfiltration is used to treat plants with modified strains of Agrobacterium tumefaciens for the purpose of transient in planta expression of genes transferred from the bacterium. These genes encode valuable recombinant proteins for therapeutic or industrial applications. Treatment of large quantities of plants for industrial-scale protein production exposes bacteria (harboring genes of interest) to agroinfiltration medium that is devoid of nutrients and carbon sources for prolonged periods of time (possibly upwards of 24 h). Such conditions may negatively influence bacterial viability, infectivity of plant cells, and target protein production. Here, we explored the role of timing in bacterial culture preparation for agroinfiltration using mass spectrometry-based proteomics to define changes in cellular processes. We observed distinct profiles associated with bacterial treatment conditions and exposure timing, including significant changes in proteins involved in pathogenesis, motility, and nutrient acquisition systems as the bacteria adapt to the new environment. These data suggest a progression towards increased cellular remodelling over time. In addition, we described changes in growth- and environment-specific processes over time, underscoring the interconnectivity of pathogenesis and chemotaxis-associated proteins with transport and metabolism. Overall, our results have important implications for the production of transiently expressed target protein products, as prolonged exposure to agroinfiltration medium suggests remodelling of the bacterial proteins towards enhanced infection of plant cells.

Transformation of TNT, 2,4-DNT, and PETN by Raoultella planticola M30b and Rhizobium radiobacter M109 and exploration of the associated enzymes.

The nitrated compounds 2,4-dinitrotoluene (2,4-DNT), 2,4,6-trinitrotoluene (TNT), and pentaerythritol tetranitrate (PETN) are toxic xenobiotics widely used in various industries. They often coexist as environmental contaminants. The aims of this study were to evaluate the transformation of 100 mg L-1 of TNT, 2,4-DNT, and PETN by Raoultella planticola M30b and Rhizobium radiobacter M109c and identify enzymes that may participate in the transformation. These strains were selected from 34 TNT transforming bacteria. Cupriavidus metallidurans DNT was used as a reference strain for comparison purposes. Strains DNT, M30b and M109c transformed 2,4-DNT (100%), TNT (100, 94.7 and 63.6%, respectively), and PETN (72.7, 69.3 and 90.7%, respectively). However, the presence of TNT negatively affects 2,4-DNT and PETN transformation (inhibition > 40%) in strains DNT and M109c and fully inhibited (100% inhibition) 2,4-DNT transformation in R. planticola M30b.Genomes of R. planticola M30b and R. radiobacter M109c were sequenced to identify genes related with 2,4-DNT, TNT or PETN transformation. None of the tested strains presented DNT oxygenase, which has been previously reported in the transformation of 2,4-DNT. Thus, unidentified novel enzymes in these strains are involved in 2,4-DNT transformation. Genes encoding enzymes homologous to the previously reported TNT and PETN-transforming enzymes were identified in both genomes. R. planticola M30b have homologous genes of PETN reductase and xenobiotic reductase B, while R. radiobacter M109c have homologous genes to GTN reductase and PnrA nitroreductase. The ability of these strains to transform explosive mixtures has a potentially biotechnological application in the bioremediation of contaminated environments.

Overexpression of polygalacturonase-inhibiting protein (PGIP) gene from Hypericum perforatum alters expression of multiple defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens in tobacco.

Hypericum perforatum L is a remarkable source of high-value secondary metabolites with increasing applications in pharmaceutical industry. However, improvement in the production of secondary metabolites through genetic engineering is a demanding task, as H. perforatum is not amenable to Agrobacterium tumefaciens-mediated transformation. In this study, we identified a Polygalacturonase-inhibiting protein (PGIP) gene from a subtractive cDNA library of A. tumefaciens-treated H. perforatum suspension cells. The role of HpPGIP in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum overexpressing HpPGIP alone or fused at the N-terminus to Phenolic oxidative coupling protein (Hyp-1), a gene that positively modulates resistance to A. tumefaciens. Furthermore, virus-induced gene silencing was employed to knock down the expression of the PGIP homologous in N. benthamiana. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in both HpPGIP and Hyp-1-PGIP transgenic plants, as assessed by GUS staining assays. However, silencing of PGIP in N. benthamiana increased the resistance to A. tumefaciens rather than susceptibility, which correlated with induction of pathogenesis-related proteins (PRs). The expression of core genes involved in several defense pathways was also analyzed in transgenic tobacco plants. Overexpression of HpPGIP led to up-regulation of key genes involved in hormone signaling, microRNA-based gene silencing, homeostasis of reactive oxygen species, and the phenylpropanoid pathway. Overexpression of Hyp-1-PGIP seemed to enhance the effect of PGIP on the expression of most genes analyzed. Moreover, HpPGIP was detected in the cytoplasm, nucleus and the plasma membrane or cell wall by confocal microscopy. Overall, our findings suggest HpPGIP modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

Agrobacterium tumefaciens ferritins play an important role in full virulence through regulating iron homeostasis and oxidative stress survival.

Ferritins are a large family of iron storage proteins, which are used by bacteria and other organisms to avoid iron toxicity and as a safe iron source in the cytosol. Agrobacterium tumefaciens, a phytopathogen, has two ferritin-encoding genes: atu2771 and atu2477. Atu2771 is annotated as a Bfr-encoding gene (Bacterioferritin, Bfr) and atu2477 as a Dps-encoding gene (DNA binding protein from starved cells, Dps). Three deletion mutants (Δbfr, Δdps, and bfr-dps double-deletion mutant ΔbdF) of these two ferritin-encoding genes were constructed to investigate the effects of ferritin deficiency on the iron homeostasis, oxidative stress resistance, and pathogenicity of A. tumefaciens. Deficiency of two ferritins affects the growth of A. tumefaciens under iron starvation and excess. When supplied with moderate iron, the growth of A. tumefaciens is not affected by the deficiency of ferritin. Deficiency of ferritin significantly reduces iron accumulation in the cells of A. tumefaciens, but the effect of Bfr deficiency on iron accumulation is severer than Dps deficiency and the double mutant ΔbdF has the least intracellular iron content. All three ferritin-deficient mutants showed a decreased tolerance to 3 mM H2 O2 in comparison with the wild type. The tumour induced by each of three ferritin-deficient mutants is less than that of the wild type. Complementation reversed the effects of ferritin deficiency on the growth, iron homeostasis, oxidative stress resistance, and tumorigenicity of A. tumefaciens. Therefore, ferritin plays an important role in the pathogenesis of A. tumefaciens through regulating iron homeostasis and oxidative stress survival.

The Presence of the Hairy-Root-Disease-Inducing (Ri) Plasmid in Wheat Endophytic Rhizobia Explains a Pathogen Reservoir Function of Healthy Resistant Plants.

A large number of strains in the Rhizobium radiobacter species complex (biovar 1 Agrobacterium) have been known as causative pathogens for crown gall and hairy root diseases. Strains within this complex were also found as endophytes in many plant species with no symptoms. The aim of this study was to reveal the endophyte variation of this complex and how these endophytic strains differ from pathogenic strains. In this study, we devised a simple but effective screening method by exploiting the high resolution power of mass spectrometry. We screened endophyte isolates from young wheat and barley plants, which are resistant to the diseases, and identified seven isolates from wheat as members of the R. radiobacter species complex. Through further analyses, we assigned five strains to the genomovar (genomic group) G1 and two strains to G7 in R. radiobacter Notably, these two genomovar groups harbor many known pathogenic strains. In fact, the two G7 endophyte strains showed pathogenicity on tobacco, as well as the virulence prerequisites, including a 200-kbp Ri plasmid. All five G1 strains possessed a 500-kbp plasmid, which is present in well-known crown gall pathogens. These data strongly suggest that healthy wheat plants are reservoirs for pathogenic strains of R. radiobacterIMPORTANCE Crown gall and hairy root diseases exhibit very wide host-plant ranges that cover gymnosperm and dicot plants. The Rhizobium radiobacter species complex harbors causative agents of the two diseases. Recently, endophyte isolates from many plant species have been assigned to this species complex. We isolated seven endophyte strains belonging to the species complex from wheat plants and revealed their genomovar affiliations and plasmid profile. The significance of this study is the finding of the genomovar correlation between the endophytes and the known pathogens, the presence of a virulence ability in two of the seven endophyte strains, and the high ratio of the pathogenic strains in the endophyte strains. This study therefore provides convincing evidence that could unravel the mechanism that maintains pathogenic agents of this species and sporadically delivers them to susceptible plants.

Quorum quenching acylase impacts the viability and morphological change of Agrobacterium tumefaciens cells.

Acylase is known as a quorum quenching enzyme that degrades N-acyl-homoserine lactones (AHLs), a key signaling molecule in a quorum sensing (QS) mechanism. Acylase I cleaves the acyl-chain in the chemical structures of AHLs, thereby exerting an anti-biofilm effect by the inhibition of bacterial cell-cell communication and resultant secretion of extracellular polymeric substances (EPS). However, the physical and physiological impacts of acylase on bacterial cells remain to be systematically elucidated. This study, therefore, investigated the effect of active and inactive acylase addition on the growth, viability, and cell morphologies of Agrobacterium tumefaciens. For comparison, active and inactive lysozymes were taken as positive controls. The results showed that active acylase inhibited A. tumefaciens cell growth at concentrations ranging from 0.1 to 1000 µg mL-1, and so did active lysozyme. Fluorescent detection by Live/Dead staining underpinned that cell viability of A. tumefaciens decreased at concentrations higher than 0.1 µg mL-1 for both acylase and lysozyme, although lysozyme inflicted higher degree of cellular damage. Moreover, atomic force microscopy unraveled a noticeable distortion of A. tumefaciens cells by both acylase and lysozyme. Together, the results showed that acylase not only blocked AHLs-based QS mechanisms but also compromised cell viability and altered surface morphology of A. tumefaciens cells, as observed by the addition of hydrolase.

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.

Acidocalcisomes and Polyphosphate Granules Are Different Subcellular Structures in Agrobacterium tumefaciens.

Acidocalcisomes are membrane-enclosed, polyphosphate-containing acidic organelles in lower Eukaryota but have also been described for Agrobacterium tumefaciens (M. Seufferheld, M. Vieira, A. Ruiz, C. O. Rodrigues, S. Moreno, and R. Docampo, J Biol Chem 278:29971-29978, 2003, https://doi.org/10.1074/jbc.M304548200). This study aimed at the characterization of polyphosphate-containing acidocalcisomes in this alphaproteobacterium. Unexpectedly, fluorescence microscopic investigation of A. tumefaciens cells using fluorescent dyes and localization of constructed fusions of polyphosphate kinases (PPKs) and of vacuolar H+-translocating pyrophosphatase (HppA) with enhanced yellow fluorescent protein (eYFP) suggested that acidocalcisomes and polyphosphate are different subcellular structures. Acidocalcisomes and polyphosphate granules were frequently located close together, near the cell poles. However, they never shared the same position. Mutant strains of A. tumefaciens with deletions of both ppk genes (Δppk1 Δppk2) were unable to form polyphosphate but still showed cell pole-located eYFP-HppA foci and could be stained with MitoTracker. In conclusion, A. tumefaciens forms polyP granules that are free of a surrounding membrane and thus resemble polyP granules of Ralstonia eutropha and other bacteria. The composition, contents, and function of the subcellular structures that are stainable with MitoTracker and harbor eYFP-HppA remain unclear.IMPORTANCE The uptake of alphaproteobacterium-like cells by ancestors of eukaryotic cells and subsequent conversion of these alphaproteobacterium-like cells to mitochondria are thought to be key steps in the evolution of the first eukaryotic cells. The identification of acidocalcisomes in two alphaproteobacterial species some years ago and the presence of homologs of the vacuolar proton-translocating pyrophosphatase HppA, a marker protein of the acidocalcisome membrane in eukaryotes, in virtually all species within the alphaproteobacteria suggest that eukaryotic acidocalcisomes might also originate from related structures in ancestors of alphaproteobacterial species. Accordingly, alphaproteobacterial acidocalcisomes and eukaryotic acidocalcisomes should have similar features. Since hardly any information is available on bacterial acidocalcisomes, this study aimed at the characterization of organelle-like structures in alphaproteobacterial cells, with A. tumefaciens as an example.

An optimized double T-DNA binary vector system for improved production of marker-free transgenic tobacco plants.

OBJECTIVES: In the plant transformation process, marker genes play a vital role in identifying transformed cells from non-transformed cells. However, once transgenic plants have been obtained, the presence of marker genes may provoke public concern about environmental or biosafety issues. In our previous study, a double T-DNA vector system has been developed to obtain marker-free transgenic plants, but the T-DNA left border (LB) and right border (RB) of the vector showed an RB-LB-RB-LB pattern and led to high linkage integration between the selectable marker gene (SMG) and the gene of interest (GOI). To improve this double T-DNA vector system, we inverted the first T-DNA direction such that a LB-RB-RB-LB pattern resulted to avoid transcriptional read-through at the LB and the subsequent linkage transfer of the SMG and GOI. RESULTS: We separately inserted the green fluorescent protein (GFP) gene as the GOI and the neomycin phosphotransferase II (NPTII) gene as the SMG in both optimized and original vectors and carried out Agrobacterium-mediated tobacco transformation. Statistical analysis revealed that the linkage frequency was 25.6% in T0 plants transformed with the optimized vector, which is a 42.1% decrease compared with that of the original vector (44.2%). The frequency of obtaining marker-free transgenic plants was 66.7% in T1 plants transformed with the optimized vector, showing a 33.4% increase compared with that of the original vector (50.0%). CONCLUSION: Our results demonstrate that the optimized double T-DNA binary vector system is a more effective, economical and time-saving approach for obtaining marker-free transgenic plants.

Perception of Agrobacterium tumefaciens flagellin by FLS2XL confers resistance to crown gall disease.

Bacterial flagella are perceived by the innate immune systems of plants1 and animals2 alike, triggering resistance. Common to higher plants is the immunoreceptor FLAGELLIN-SENSING 2 (FLS2)3, which detects flagellin via its most conserved epitope, flg22. Agrobacterium tumefaciens, which causes crown gall disease in many crop plants, has a highly diverged flg22 epitope and evades immunodetection by plants so far studied. We asked whether, as a next step in this game of 'hide and seek', there are plant species that have evolved immunoreceptors with specificity for the camouflaged flg22Atum of A. tumefaciens. In the wild grape species Vitis riparia, we discovered FLS2XL, a previously unknown form of FLS2, that provides exquisite sensitivity to typical flg22 and to flg22Atum. As exemplified by ectopic expression in tobacco, FLS2XL can limit crown gall disease caused by A. tumefaciens.

Embryonic Explant and Plumular Meristem Transformation Methods for Development of Transgenic Pigeon Pea.

A reliable pigeon pea transformation system can assist the rapid improvement of this important grain legume through transgenic development. Here we describe two methods of Agrobacterium tumefaciens-mediated pigeon pea transformation. In the tissue culture based embryonic explant transformation method, microshoot grafting was included to obtain rapid root induction, while the other method was culture independent and designated as plumular meristem transformation. Both methods drastically enhanced the transformation frequency and have the potential to provide reasonable solutions for maximum transgenic recovery in biotechnological breeding programs.

ABC transporter genes ABC-C6 and ABC-G33 alter plant-microbe-parasite interactions in the rhizosphere.

Plants are master regulators of rhizosphere ecology, secreting a complex mixture of compounds into the soil, collectively termed plant root exudate. Root exudate composition is highly dynamic and functional, mediating economically important interactions between plants and a wide range of soil organisms. Currently we know very little about the molecular basis of root exudate composition, which is a key hurdle to functional exploitation of root exudates for crop improvement. Root expressed transporters modulate exudate composition and could be manipulated to develop beneficial plant root exudate traits. Using Virus Induced Gene silencing (VIGS), we demonstrate that knockdown of two root-expressed ABC transporter genes in tomato cv. Moneymaker, ABC-C6 and ABC-G33, alters the composition of semi-volatile compounds in collected root exudates. Root exudate chemotaxis assays demonstrate that knockdown of each transporter gene triggers the repulsion of economically relevant Meloidogyne and Globodera spp. plant parasitic nematodes, which are attracted to control treatment root exudates. Knockdown of ABC-C6 inhibits egg hatching of Meloidogyne and Globodera spp., relative to controls. Knockdown of ABC-G33 has no impact on egg hatching of Meloidogyne spp. but has a substantial inhibitory impact on egg hatching of G. pallida. ABC-C6 knockdown has no impact on the attraction of the plant pathogen Agrobacterium tumefaciens, or the plant growth promoting Bacillus subtilis, relative to controls. Silencing ABC-G33 induces a statistically significant reduction in attraction of B. subtilis, with no impact on attraction of A. tumefaciens. By inoculating selected differentially exuded compounds into control root exudates, we demonstrate that hexadecaonic acid and pentadecane are biologically relevant parasite repellents. ABC-C6 represents a promising target for breeding or biotechnology intervention strategies as gene knockdown leads to the repulsion of economically important plant parasites and retains attraction of the beneficial rhizobacterium B. subtilis. This study exposes the link between ABC transporters, root exudate composition, and ex planta interactions with agriculturally and economically relevant rhizosphere organisms, paving the way for new approaches to rhizosphere engineering and crop protection.