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Crown gall caused by Agrobacterium vitis (A. vitis) is one of the crucial issues restricting the to grape industry. In this study, Agrobacterium tumefaciens (Ag-8) was separated from the soil that could prevent the occurrence of grape crown gall. By the mutagenesis of Ag-8 transposon, the siaD gene deletion strain (ΔsiaD) showed significantly lower efficacy in grape and tomato plants for controlling grape crown gall, but the relevant mechanism was not clear. The biofilm formation and motility of ΔsiaD were significantly decreased, and the colonization ability of ΔsiaD in tomato roots was significantly reduced. RNA-seq analysis showed that the expression of nemR significantly reduced in the ΔsiaD and that the expression of nemR showed a high correlation with biofilm and motility. Further studies showed that the nemR gene deletion strain of Ag-8 (ΔnemR) showed significantly reduced motility, biofilm formation and control of grape crown gall compared to Ag-8, and the nemR gene complementary strain of Ag-8 (ΔnemR-comp) recovered to Ag-8 wild-type levels. The inoculation experiments of preventive, curative or simultaneous treatment further showed that the preferential inoculation with Ag-8 reduced the incidence of grape crown gall on tomato plants, and studies showed that the mutation of siaD affected the site competition between Ag-8 and A. vitis, and that the mutation of nemR was consistent with the previous results. This study provides a new strategy for the prevention and control of grape crown gall, which is of great significance to the grape industry to increase production and income., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

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 β-lactamase production, a response dependent on the broad-spectrum β-lactamase AmpC and its transcription factor, AmpR. Loss of the putative ampD orthologue atu2113 led to constitutive production of AmpC-dependent β-lactamase activity and ampicillin resistance. Finally, one cell wall remodeling enzyme, MltB3, was necessary for the AmpC-dependent β-lactamase activity, and its loss elicited ampicillin and carbenicillin sensitivity in the A. tumefaciens C58 and GV3101 strains. Furthermore, GV3101 Δ mltB3 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 β-lactam resistance in A. tumefaciens. Knowledge about proteins that promote or inhibit β-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.

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 β-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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2021 John Wiley & Sons Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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 10 6 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 10 6 spores for the hisB marker and 318 transformants per 10 6 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.

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., (© 2021 John Wiley & Sons Ltd.)

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.

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.

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., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

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 H 2 O 2 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., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

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. radiobacter IMPORTANCE 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., (Copyright © 2020 American Society for Microbiology.)

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., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 American Society for Microbiology.)

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 T 0 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 T 1 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.

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.

Bacterial flagella are perceived by the innate immune systems of plants 1 and animals 2 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 flg22 Atum of A. tumefaciens. In the wild grape species Vitis riparia, we discovered FLS2 XL , a previously unknown form of FLS2, that provides exquisite sensitivity to typical flg22 and to flg22 Atum . As exemplified by ectopic expression in tobacco, FLS2 XL can limit crown gall disease caused by A. tumefaciens.

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.

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.

Antarctica, being the coldest, driest, and windiest continent on Earth, represents the most extreme environment in which a living organism can survive. Under constant exposure to harsh environmental threats, terrestrial Antarctica remains home to a great diversity of microorganisms, indicating that the soil bacteria must have adapted a range of survival strategies that require cell-to-cell communication. Survival strategies include secondary metabolite production, biofilm formation, bioluminescence, symbiosis, conjugation, sporulation, and motility, all of which are often regulated by quorum sensing (QS), a type of bacterial communication. Until now, such mechanisms have not been explored in terrestrial Antarctica. In this study, LuxI/LuxR-based quorum sensing (QS) activity was delineated in soil bacterial isolates recovered from Adams Flat, in the Vestfold Hills region of East Antarctica. Interestingly, we identified the production of potential homoserine lactones (HSLs) with chain lengths ranging from medium to long in 19 bacterial species using three biosensors, namely, Agrobacterium tumefaciens NTL4, Chromobacterium violaceum CV026, and Escherichia coli MT102, in conjunction with thin-layer chromatography (TLC). The majority of detectable HSLs were from Gram-positive species not previously known to produce HSLs. This discovery further expands our understanding of the microbial community capable of this type of communication, as well as provides insights into physiological adaptations of microorganisms that allow them to survive in the harsh Antarctic environment. IMPORTANCE Quorum sensing, a type of bacterial communication, is widely known to regulate many processes, including those that confer a survival advantage. However, little is known about communication by bacteria residing within Antarctic soils. Employing a combination of bacterial biosensors, analytical techniques, and genome mining, we found a variety of Antarctic soil bacteria speaking a common language, via LuxI/LuxR-based quorum sensing, thus potentially supporting survival in a mixed microbial community. This study reports potential quorum sensing activity in Antarctic soils and has provided a platform for studying physiological adaptations of microorganisms that allow them to survive in the harsh Antarctic environment., (Copyright © 2019 American Society for Microbiology.)

Agrobacterium-mediated plant galls are often misdiagnosed as nematode-mediated knots, even by experts, because the gall symptoms in both conditions are very similar. In the present study, we developed biosensor strains based on agrobacterial opine metabolism that easily and simply diagnoses Agrobacterium-induced root galls. Our biosensor consists of Agrobacterium mannitol (ABM) agar medium, X-gal, and a biosensor. The working principle of the biosensor is that exogenous nopaline produced by plant root galls binds to NocR, resulting in NocR/nopaline complexes that bind to the promoter of the nopaline oxidase gene (nox) operon and activate the transcription of noxB-lacZY, resulting in readily visualized blue pigmentation on ABM agar medium supplemented with X-gal (ABMX-gal). Similarly, exogenous octopine binds to OccR, resulting in OoxR/octopine complexes that bind to the promoter of the octopine oxidase gene (oox) operon and activate the transcription of ooxB-lacZY, resulting in blue pigmentation in the presence of X-gal. Our biosensor is successfully senses opines produced by Agrobacterium-infected plant galls, and can be applied to easily distinguish Agrobacterium crown gall disease from nematode disease.

Main Conclusion: Phenolic oxidative coupling protein (Hyp-1) isolated from Hypericum perforatum L. was characterized as a defense gene involved in H. perforatum recalcitrance to Agrobacterium tumefaciens-mediated transformation Hypericum perforatum L. is a reservoir of high-value secondary metabolites of increasing interest to researchers and to the pharmaceutical industry. However, improving their production via genetic manipulation is a challenging task, as H. perforatum is recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, phenolic oxidative coupling protein (Hyp-1), a pathogenesis-related (PR) class 10 family gene, was selected from a subtractive cDNA library from A. tumefaciens-treated H. perforatum suspension cells. The role of Hyp-1 in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum and Lactuca sativa overexpressing Hyp-1, and in Catharanthus roseus silenced for its homologous Hyp-1 gene, CrIPR. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in Hyp-1 transgenic plants. However, silencing of CrIPR induced CrPR-5 expression and decreased expression efficiency of Agrobacterium. The expression of core genes involved in several defense pathways was also analyzed in Hyp-1 transgenic tobacco plants. Overexpression of Hyp-1 led to an ample down-regulation of key genes involved in auxin signaling, microRNA-based gene silencing, detoxification of reactive oxygen species, phenylpropanoid pathway and PRs. Moreover, Hyp-1 was detected in the nucleus, plasma membrane and the cytoplasm of epidermal cells by confocal microscopy. Overall, our findings suggest Hyp-1 modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

In this study, we explored the pathogenicity and phylogenetic position of Agrobacterium spp. strains isolated from crown gall tissues on annual, perennial, and ornamental plants in Iran. Of the 43 strains studied, 10 strains were identified as Allorhizobium vitis (formerly Agrobacterium vitis ) using the species-specific primer pair PGF/PGR. Thirty-three remaining strains were studied using multilocus sequence analysis of four housekeeping genes (i.e., atpD , gyrB , recA , and rpoB ), from which seven strains were identified as A. larrymoorei and one strain was identified as A. rubi (Rer); the remaining 25 strains were scattered within the A. tumefaciens species complex. Two strains were identified as genomospecies 1 (G1), seven strains were identified as A. radiobacter (G4), seven strains were identified as A. deltaense (G7), two strains were identified as A. nepotum (G14), and one strain was identified as " A. viscosum " (G15). The strains Rnr, Rnw, and Rew as well as the two strains OT33 and R13 all isolated from rose and the strain Ap1 isolated from apple were clustered in three atypical clades within the A. tumefaciens species complex. All but eight strains (i.e., Nec10, Ph38, Ph49, fic9, Fic72, R13, OT33, and Ap1) were pathogenic on tomato and sunflower seedlings in greenhouse conditions, whereas all but three strains (i.e., fic9, Fic72, and OT33) showed tumorigenicity on carrot root discs. The phylogenetic analysis and nucleotide diversity statistics suggested the existence of two novel genomospecies within the A. tumefaciens species complex, which we named "G19" and "G20." Hence, we propose the strains Rew, Rnw, and Rnr as the members of "G19" and the strains R13 and OT33 as the members of G20, whereas the phylogenetic status of the atypical strain Ap1 remains undetermined.

The Agrobacterium tumefaciens ActSR two-component regulatory system is a member of a homologous group of global redox-responsive regulatory systems that adjust the expression of energy-consuming and energy-supplying metabolic pathways in order to maintain cellular redox balance. In this study, the transcriptional organization of the hrpB-actSR locus was determined and the effect of actSR system inactivation on stress resistance was investigated. It was found that hrpB is transcribed as a monocistronic mRNA and actS is transcribed along with actR as a bicistronic mRNA, while actR is also transcribed as a monocistronic message. Each message is initiated from a separate promoter. Inactivation of actR resulted in decreased resistance to membrane stress (sodium dodecyl sulfate), acid stress (pH 5.5), iron starvation (bipyridyl) and iron excess (FeCl 3 ), and antibiotic stress (tetracycline and ciprofloxacin). Resistance to oxidative stress in the form of organic peroxide (cumene hydroperoxide) increased, while resistance to inorganic peroxide (H 2 O 2 ) decreased. An actR insertion mutant displayed reduced catalase activity, even though transcription of katA and catE remained unchanged. Complementation of the actR inactivation mutant with plasmid-encoded actR or overexpression of hemH , encoding ferrochelatase, restored wild-type catalase activity and H 2 O 2 resistance levels. Gel mobility shift and hemH promoter -lacZ fusion results indicated that ActR is a positive regulator of hemH that binds directly to the hemH promoter region. Thus, inactivation of the A. tumefaciens ActSR system affects resistance to multiple stresses, including reduced resistance to H 2 O 2 resulting from a reduction in catalase activity due to reduced expression of hemH .

The fungus causing target spot disease, Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei, poses an increasing threat to watermelon (Citrullus lanatus), muskmelon (Cucumis melo), and cucumber (Cucumis sativus); the most economically important cucurbit crops grown in China. An understanding of the molecular mechanisms underlying the pathogenicity of C. cassiicola is essential for the development of new strategies to control this disease-causing fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) might be useful to obtain transformants of C. cassiicola, for the ultimate identification of genes involved in pathogenicity. In the present work, we established and optimized an ATMT protocol using A. tumefaciens strain AGL-1 carrying the vector pATMT1 for C. cassiicola. Efficiency of ATMT was 102-148 transformants per 10 6 conidia and successive subculturing of transformants on non-selective and selective media demonstrated that the integrated transfer (T)-DNA was stably inherited in C. cassiicola transformants. The integration of the hygromycin B phosphotransferase (hph) gene into C. cassiicola was validated by PCR and Southern blot analyses, which revealed that nearly 90 % of the transformants contained single-copy T-DNA. The transformants with altered phenotypes were characterized. Three of these transformants completely lost pathogenicity and other three showed strongly impaired pathogenicity relative to the Cc-GX strain on muskmelon leaves. These results strongly suggest that ATMT may be used as a molecular tool for identifying genes relevant to pathogenicity in the fungus C. cassiicola, an emerging threat to several agronomically important plants in China., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The type VI secretion system (T6SS) is used by gram-negative bacteria to translocate effectors that can antagonize other bacterial cells. Models predict the variation in collections of effector and cognate immunity genes determine competitiveness and can affect the dynamics of populations and communities of bacteria. However, the outcomes of competition cannot be entirely explained by compatibility of effector-immunity (EI) pairs. Here, we characterized the diversity of T6SS loci of plant-pathogenic Agrobacterium tumefaciens and showed that factors other than EI pairs can impact interbacterial competition. All examined strains encode T6SS active in secretion and antagonism against Escherichia coli . The spectra of EI pairs as well as compositions of gene neighborhoods are diverse. Almost 30 in-planta competitions were tested between different genotypes of A. tumefaciens . Fifteen competitions between members of different species-level groups resulted in T6SS-dependent suppression in in-planta growth of prey genotypes. In contrast, ten competitions between members within species-level groups resulted in no significant effect on the growth of prey genotypes. One strain was an exceptional case and, despite encoding a functional T6SS and toxic effector protein, could not compromise the growth of the four tested prey genotypes. The data suggest T6SS-associated EI pairs can influence the competitiveness of strains of A. tumefaciens , but genetic features have a significant role on the efficacy of interbacterial antagonism.

Crown gall disease caused by Agrobacterium tumefaciens severely impacts the production of peach and other fruit trees. Several peach cultivars are partially resistant to A. tumefaciens , but little is known about the roles of endophytic microbiota in disease resistance. In the present study, the endophytic bacterial communities of resistant and susceptible peach cultivars "Honggengansutao" and "Okinawa" were analyzed using universal 16S rRNA gene amplicon sequencing in parallel with the cultivation and characterization of bacterial isolates. A total of 1,357,088 high-quality sequences representing 3,160 distinct operational taxonomic units (OTUs; Proteobacteria , Actinobacteria , Bacteroidetes , and Firmicutes ) and 1,200 isolates of 20 genera and 305 distinct ribotypes were collected from peach roots and twigs. It was found that factors including plant developmental stage, cultivar, and A. tumefaciens invasion strongly influenced the peach endophytic communities. The community diversity of endophytic bacteria and the abundance of culturable bacteria were both higher in the roots of the resistant cultivar, particularly after inoculation. Strikingly, the pathogen antagonists Streptomyces and Pseudomonas in roots and Rhizobium in twigs were most frequently detected in resistant plants. Our results suggest that the higher abundance and diversity of endophytic bacteria and increased proportions of antagonistic bacteria might contribute to the natural defense of the resistant cultivar against A. tumefaciens This work reveals the relationships between endophytic bacteria and disease resistance in peach plants and provides important information for microbiome-based biocontrol of crown gall disease in fruit trees. IMPORTANCE Agrobacterium tumefaciens as the causal agent of peach crown gall disease can be controlled by planting resistant cultivars. This study profiles the endophytic bacteria in susceptible and resistant peach cultivars, advancing our understanding of the relationships between endophytic bacterial communities and peach crown gall disease, with potential implications for other complex microbiome-plant-pathogen interactions. The resistant cultivar may defend itself by increasing the diversity and abundance of beneficial endophytic bacteria. The antagonists identified among the genera Streptomyces , Pseudomonas , and Rhizobium may have application potential for biocontrol of crown gall disease in fruit trees., (Copyright © 2019 American Society for Microbiology.)

Agrobacterium tumefaciens is a niche-constructing biotroph that exploits host plant metabolites. We combined metabolomics, transposon-sequencing (Tn-seq), transcriptomics, and reverse genetics to characterize A. tumefaciens pathways involved in the exploitation of resources from the Solanum lycopersicum host plant. Metabolomics of healthy stems and plant tumors revealed the common (e.g. sucrose, glutamate) and enriched (e.g. opines, γ-aminobutyric acid (GABA), γ-hydroxybutyric acid (GHB), pyruvate) metabolites that A. tumefaciens could use as nutrients. Tn-seq and transcriptomics pinpointed the genes that are crucial and/or upregulated when the pathogen grew on either sucrose (pgi, kdgA, pycA, cisY) or GHB (blcAB, pckA, eno, gpsA) as a carbon source. While sucrose assimilation involved the Entner-Doudoroff and tricarboxylic acid (TCA) pathways, GHB degradation required the blc genes, TCA cycle, and gluconeogenesis. The tumor-enriched metabolite pyruvate is at the node connecting these pathways. Using reverse genetics, we showed that the blc, pckA, and pycA loci were important for aggressiveness (tumor weight), proliferation (bacterial charge), and/or fitness (competition between the constructed mutants and wild-type) of A. tumefaciens in plant tumors. This work highlighted how a biotroph mobilizes its central metabolism for exploiting a wide diversity of resources in a plant host. It further shows the complementarity of functional genome-wide scans by transcriptomics and Tn-seq to decipher the lifestyle of a plant pathogen., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Wheat is the most widely grown staple food crop in the world and accounts for dietary needs of more than 35% of the human population. Current status of transgenic wheat development is slow all over the world due to the lack of a suitable transformation system. In the present study, an efficient and reproducible Agrobacterium-mediated transformation system in bread wheat (Triticum aestivum L.) is established. The mature and immature embryos of six recently released high yielding spring bread wheat genotypes were used to standardize various parameters using Agrobacterium tumefaciens strain EHA105 harbouring binary vector pCAMBIA3301 having gus and bar as marker genes. The optimum duration for embryo pre-culture, inoculation time and co-cultivation were 2 days, 30 min and 48 h, respectively. The bacterial inoculum concentration of OD of 1 at 600 nm showed 67.25% transient GUS expression in the histochemical GUS assay. The filter paper based co-cultivation limits the Agrobacterium overgrowth and had 82.3% explants survival rate whereas medium based strategy had 22.7% explants survival only. The medium having picloram 4 mg/l along with antibiotics (cefotaxime 500 mg/l and timentin 300 mg/l) was found best suitable for initial week callus induction. The standardized procedure gave overall 14.9% transformation efficiency in immature embryos and 9.8% in mature embryos and confirmed by gene-specific and promoter-specific PCR and southern analysis. These results indicate that the developed Agrobacterium-mediated transformation system is suitable for diverse wheat genotypes. The major obstacle for the implication of the CRISPR-based genome editing techniques is the non-availability of a suitable transformation system. Thus, the present system can be exploited to deliver the T-DNA into the wheat genome for CRISPR-based target modifications and transgene insertions.

A core regulatory pathway that directs developmental transitions and cellular asymmetries in Agrobacterium tumefaciens involves two overlapping, integrated phosphorelays. One of these phosphorelays putatively includes four histidine sensor kinase homologues, DivJ, PleC, PdhS1 and PdhS2, and two response regulators, DivK and PleD. In several different alphaproteobacteria, this pathway influences a conserved downstream phosphorelay that ultimately controls the phosphorylation state of the CtrA master response regulator. The PdhS2 sensor kinase reciprocally regulates biofilm formation and swimming motility. In the current study, the mechanisms by which the A. tumefaciens sensor kinase PdhS2 directs this regulation are delineated. PdhS2 lacking a key residue implicated in phosphatase activity is markedly deficient in proper control of attachment and motility phenotypes, whereas a kinase-deficient PdhS2 mutant is only modestly affected. A genetic interaction between DivK and PdhS2 is revealed, unmasking one of several connections between PdhS2-dependent phenotypes and transcriptional control by CtrA. Epistasis experiments suggest that PdhS2 may function independently of the CckA sensor kinase, the cognate sensor kinase for CtrA, which is inhibited by DivK. Global expression analysis of the pdhS2 mutant reveals a restricted regulon, most likely functioning through CtrA to separately control motility and regulate the levels of the intracellular signal cyclic diguanylate monophosphate (cdGMP), thereby affecting the production of adhesive polysaccharides and attachment. We hypothesize that in A. tumefaciens the CtrA regulatory circuit has expanded to include additional inputs through the addition of PdhS-type sensor kinases, likely fine-tuning the response of this organism to the soil microenvironment.

Nine transformants of Rhodosporidium toruloides with significant changes in the carotenoid profile were obtained by Agrobacterium tumefaciens-mediated transformation, including a white, three red, and four yellow mutants. A red mutant A1-15-BRQ that showed a high torularhodin production was selected for culture condition optimization. Results indicated that the torularhodin yield was boosted with glucose as the carbon source, at a carbon/nitrogen ratio of 22, a loading volume of 75 mL, and 28 °C. The torularhodin yield of 21.3 mg/L consisting of 94.4% total carotenoids was obtained by Box-Behnken design experiments. The torularhodin yield was 17.0-fold higher than that of the wild type, with time shortened from 9 to 3 days. This study reports an effective strategy for improving torularhodin production and provides a candidate R. toruloides strain for highly selective production of torularhodin.

Several approaches have recently been adopted to improve Agrobacterium -mediated transformation of maize; however, about eight months of in vitro culture are still required to isolate transgenic plants. Furthermore, genetic transformation of maize depends on immature embryos, which greatly increases costs. Here, we report a method that ensures the competency of an embryogenic callus secondary culture under laboratory conditions for Agrobacterium -mediated transformation. Moreover, pretreatment of the cell wall with a mixed lytic enzyme solution prior to Agrobacterium infection, significantly improved transformation efficiency and stability. Average stable transformation efficiency was approximately 30.39%, with peaks of 94.46%. Expression and phenotypic analysis of the Rsc reporter gene were tested in the T₀ generation of transgenic plants. Using this system, we successfully regenerated transgenic maize plantlets within three months of the emergence of the embryogenic callus. Additionally, we reduced somaclonal variation accompanying prolonged culture of maize cells in the dedifferentiated state, thus facilitating the molecular breeding of maize.

Agrobacterium tumefaciens is a plant pathogen which provokes galls on roots and stems (crown-gall disease) and colonizes them. Two approaches combining omics were used to decipher the lifestyle of A. tumefaciens in plant tumors: an integrative approach when omics were used on A. tumefaciens cells collected from plant tumors, a deconvolution approach when omics were used on A. tumefaciens cells exploiting a single tumor metabolite in pure culture assay. This addendum highlights some recent results on the biotroph lifestyle of A. tumefaciens in plant tumors.

Acinetobacter baumannii cell-cell signaling is mediated by production and release of N-acyl homoserine lactones (AHLs), primarily N-(3-hydroxydodecanoyl)-L-homoserine lactone (3-hydroxy-C 12 -HSL). The secretion of this signal can be readily assessed via simple plate-based or thin-layer chromatography-based assays utilizing an Agrobacterium tumefaciens traG-lacZ containing biosensor. In this chapter, we describe methods to assay for secreted N-acyl homoserine lactone production in Acinetobacter baumannii.

We previously described a plasmid of Agrobacterium spp., pAoF64/95, in which the quorum-sensing system that controls conjugative transfer is induced by the opine mannopine. We also showed that the quorum-sensing regulators TraR, TraM, and TraI function similarly to their counterparts in other repABC plasmids. However, traR, unlike its counterpart on Ti plasmids, is monocistronic and not located in an operon that is inducible by the conjugative opine. Here, we report that both traR and traM are expressed constitutively and not regulated by growth with mannopine. We report two additional regulatory genes, mrtR and tmsP, that are involved in a novel mechanism of control of TraR activity. Both genes are located in the distantly linked region of pAoF64/95 encoding mannopine utilization. MrtR, in the absence of mannopine, represses the four-gene mocC operon as well as tmsP, which is the distal gene of the eight-gene motA operon. As judged by a bacterial two-hybrid analysis, TmsP, which shows amino acid sequence relatedness with the TraM-binding domain of TraR, interacts with the antiactivator. We propose a model in which mannopine, acting through the repressor MrtR, induces expression of TmsP which then titrates the levels of TraM thereby freeing TraR to activate the tra regulon., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Imbalanced potassium (K) fertilization in agricultural fields has led to considerable negative impacts and remains to be the foremost challenge for maize production in India-Gangetic region. Plant growth-promoting rhizobacteria, particularly potassium solubilizing rhizobacteria (KSR), could serve as inoculants and a promising strategy for enhancement of plant absorption of K hence reducing dependency on chemical fertilizers. Maize seeds were microbiolized for 30 min with KSR suspensions. In the present study, the use of chemical fertilizers along with Agrobacterium tumefaciens strain OPVS10 showed pronounced beneficial effect on growth and yield attributes in maize. There was a significant difference among different parameters studied when varying doses of K and KSR strains were applied. Results showed that the combined application of KSR strain OPVS10 with 100% RDK (recommended dose of K) was most effective in modulating growth, physio-biochemical, and yield attributes in maize thus could be regarded as a promising alternative to mineral K-fertilization. Principal component analysis (PCA) revealed that 100-grain weight and grain yield were the most important properties to improve the sustainable growth of maize. Therefore, these KSR strains have different mechanisms for modulating various activities in maize plants. Results suggested that the synergistic application of KSR strain OPVS10 with 100% RDK can be used for optimized breeding, screening, and nutrient assimilation in maize crop. Hence, this eco-friendly approach may be one of the efficient methods for reducing dependency on chemicals, which pose adverse effects on human health directly and indirectly.

Agrobacterium-mediated transient expression is a powerful analysis platform for diverse plant gene functional studies, but the mechanisms regulating the expression or transformation levels are poorly studied. Previously, we developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST, for Arabidopsis seedlings. In this study, we found that AGROBEST could promote the growth of agrobacteria as well as inhibit the host immunity response. When the factor of agrobacterial growth is minimized, maintaining pH at 5.5 with MES buffer was the key to achieving optimal transient expression efficiency. The expression of plant immunity marker genes, FRK1 and NHL10, was suppressed in the pH-buffered medium as compared with non-buffered conditions in Col-0 and an efr-1 mutant lacking the immunity receptor EFR recognizing EF-Tu, a potent pathogen- or microbe-associated molecular pattern (PAMP or MAMP) of A. tumefaciens. Notably, such immune suppression could also occur in Arabidopsis seedlings without Agrobacterium infection. Furthermore, the PAMP-triggered influx of calcium ions was compromised in the pH-buffered medium. We propose that the enhanced transient expression efficiency by stable pH was due to inhibiting calcium ion uptake and subsequently led to suppressing immunity against Agrobacterium.

Rotary flagella propel bacteria through liquid and across semisolid environments. Flagella are composed of the basal body that constitutes the motor for rotation, the curved hook that connects to the basal body, and the flagellar filament that propels the cell. Flagellar filaments can be composed of a single flagellin protein, such as in Escherichia coli , or made up of multiple flagellins, such as in Agrobacterium tumefaciens The four distinct flagellins FlaA, FlaB, FlaC, and FlaD produced by wild-type A. tumefaciens are not redundant in function but have specific properties. FlaA and FlaB are much more abundant than FlaC and FlaD and are readily observable in mature flagellar filaments, when either FlaA or FlaB is fluorescently labeled. Cells producing FlaA with any one of the other three flagellins can generate functional filaments and thus are motile, but FlaA alone cannot constitute a functional filament. In flaA mutants that manifest swimming deficiencies, there are multiple ways by which these mutations can be phenotypically suppressed. These suppressor mutations primarily occur within or upstream of the flaB flagellin gene or in the transcription factor sciP regulating flagellin expression. The helical conformation of the flagellar filament appears to require a key asparagine residue present in FlaA and absent in other flagellins. However, FlaB can be spontaneously mutated to render helical flagella in the absence of FlaA, reflecting their overall similarity and perhaps the subtle differences in the specific functions they have evolved to fulfill. IMPORTANCE Flagellins are abundant bacterial proteins comprising the flagellar filaments that propel bacterial movement. Several members of the alphaproteobacterial group express multiple flagellins, in contrast to model systems, such as with Escherichia coli , which has one type of flagellin. The plant pathogen Agrobacterium tumefaciens has four flagellins, the abundant and readily detected FlaA and FlaB, and lower levels of FlaC and FlaD. Mutational analysis reveals that FlaA requires at least one of the other flagellins to function, as flaA mutants produce nonhelical flagella and cannot swim efficiently. Suppressor mutations can rescue this swimming defect through mutations in the remaining flagellins, including structural changes imparting helical shape to the flagella, and putative regulators. Our findings shed light on how multiple flagellins contribute to motility., (Copyright © 2018 American Society for Microbiology.)

The gene for Kunitz peptidase inhibitor-like protein (KPILP) contains nested alternative open reading frame (aORF) that controls expression of the maternal mRNA. The content of NbKPILP mRNA in intact leaves of Nicotiana benthamiana plant is low but increases significantly upon extended dark exposure or when foreign nucleic acid is overexpressed in the cells. The NbKPILP gene promoter along with the expressed nested aORF are likely to play an important role in maintaining the levels of NbKPILP mRNA. To elucidate the role of NbKPILP promoter, we isolated a fragment of N. benthamiana chromosomal DNA upstream of the NbKPILP transcription start, sequenced it, and created constructs in which reporter E. coli uidA gene coding for β-D-glucuronidase (GUS) was placed under control of the NbKPILP promoter. By assessing the efficacy of uidA mRNA synthesis directed by the NbKPILP promoter and 35S promoter of the cauliflower mosaic virus in a transient expression system, we showed that the levels of GUS accumulation were comparable for both promoters. Prolonged incubation of the agroinjected plants in the darkness stimulated accumulation of the uidA mRNA directed by the NbKPILP promoter. Our experiments indicate that along with regulation at the transcriptional level, expression of NbKPILP mRNA can be affected by expression of the nested aORF controlled by the polypurine block (PPB) located upstream of its start codon, since introduction of mutations in the PPB resulted in significant accumulation of the NbKPILP mRNA. Nucleotide replacement in the aORF start codon led to the drastic increase in the amounts of NbKPILP mRNA and its protein product.

Agrobacterium tumefaciens infects and causes crown galls in dicot plants by transferring T-DNA from the Ti plasmid to the host plant via a type IV secretion system. This process requires appropriate environmental conditions, certain plant secretions, and bacterial regulators. In our previous work, a member of the LysR family of transcriptional regulators (LsrB) in Sinorhizobium meliloti was found to modulate its symbiotic interactions with the host plant alfalfa. However, the function of its homolog in A. tumefaciens remains unclear. In this study, we show that the LsrB protein of A. tumefaciens is required for efficient transformation of host plants. A lsrB deletion mutant of A. tumefaciens exhibits a number of defects, including in succinoglycan production, attachment, and resistance to oxidative stress and iron limitation. RNA-sequencing analysis indicated that 465 genes were significantly differentially expressed (upregulation of 162 genes and downregulation of 303 genes) in the mutant, compared with the wild-type strain, including those involved in succinoglycan production, iron transporter, and detoxification enzymes for oxidative stress. Moreover, expression of the lsrB gene from S. meliloti, Brucella abortus, or A. tumefaciens rescued the defects observed in the S. meliloti or A. tumefaciens lsrB deletion mutant. Our findings suggest that a conserved mechanism of LsrB function exists in symbiotic and pathogenic bacteria of the family Rhizobiaceae.

Agrobacterium tumefaciens constructs an ecological niche in its host plant by transferring the T-DNA from its Ti plasmid into the host genome and by diverting the host metabolism. We combined transcriptomics and genetics for understanding the A. tumefaciens lifestyle when it colonizes Arabidopsis thaliana tumors. Transcriptomics highlighted: a transition from a motile to sessile behavior that mobilizes some master regulators (Hfq, CtrA, DivK and PleD); a remodeling of some cell surface components (O-antigen, succinoglucan, curdlan, att genes, putative fasciclin) and functions associated with plant defense (Ef-Tu and flagellin pathogen-associated molecular pattern-response and glycerol-3-phosphate and nitric oxide signaling); and an exploitation of a wide variety of host resources, including opines, amino acids, sugars, organic acids, phosphate, phosphorylated compounds, and iron. In addition, construction of transgenic A. thaliana lines expressing a lactonase enzyme showed that Ti plasmid transfer could escape host-mediated quorum-quenching. Finally, construction of knock-out mutants in A. tumefaciens showed that expression of some At plasmid genes seemed more costly than the selective advantage they would have conferred in tumor colonization. We provide the first overview of A. tumefaciens lifestyle in a plant tumor and reveal novel signaling and trophic interplays for investigating host-pathogen interactions., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

CLAVATA (CLV) system including CLV1-like kinase and CLE-peptides is the part of the AON (autoregulation of nodulation) that controls nodule number in legume plants. Moreover, CLV system plays a key role in meristems, where it regulates the expression of WOX genes in organizing centers. Recently, we found that WOX5 homolog in pea is also expressed in nodules and in tumors induced by Agrobacterium tumefaciens. Based on this, we hypothesized that both nodules and agrobacterial tumors may be regulated by and may trigger the same components of AON, including the same WOX and CLV genes. Here, we found that pea plants with agrobacterial tumors induced prior to rhizobial inoculation had reduced the number of nodules. This effect was absent in pea sym29 mutant defective in CLV1-like kinase, the key component of AON. That suggests that agrobacterial tumors may produce a signal activating CLV1-like kinase and thereby decrease the nodule number. Since CLE peptides are known to act upstream of CLV1-like kinase, expression analysis of CLE genes has been performed both in developing nodules and tumors. Overall, 45 CLE genes were identified, and among them nine nodulation-induced CLEs were found in pea. In agrobacterial tumors, no expression of nodule-specific CLE genes the homologues of which inhibit nodulation in other legumes was observed. However, increased expression of two other nodulation-induced CLE genes was observed in agrobacterial tumors, suggesting that CLE genes are expressed in tumors that may still contribute to autoregulatory processes suppressing nodulation., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The whitefly-transmitted geminiviruses induce severe developmental abnormalities in plants. Geminivirus-encoded C4 protein functions as one of viral symptom determinants that could induce abnormal cell division. However, the molecular mechanism by which C4 contributes to cell division induction remains unclear. Here we report that tomato leaf curl Yunnan virus (TLCYnV) C4 interacts with a glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinase, designed NbSKη, in Nicotiana benthamiana. Pro32, Asn34 and Thr35 of TLCYnV C4 are critical for its interaction with NbSKη and required for C4-induced typical symptoms. Interestingly, TLCYnV C4 directs NbSKη to the membrane and reduces the nuclear-accumulation of NbSKη. The relocalization of NbSKη impairs phosphorylation dependent degradation on its substrate-Cyclin D1.1 (NbCycD1;1), thereby increasing the accumulation level of NbCycD1;1 and inducing the cell division. Moreover, NbSKη-RNAi, 35S::NbCycD1;1 transgenic N. benthamiana plants have the similar phenotype as 35S::C4 transgenic N. benthamiana plants on callus-like tissue formation resulted from abnormal cell division induction. Thus, this study provides new insights into mechanism of how a viral protein hijacks NbSKη to induce abnormal cell division in plants.

Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due to variations in cell competency to induce embryogenic callus, as well as the cell's ability to receive and incorporate transgenes into the genome. This chapter describes two transformation protocols using Agrobacterium- and biolistic-mediated methods for gene delivery. Immature zygotic embryos of maize inbred B104, excised from ears harvested 10-14 days post pollination, are used as starting explant material. Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-Rad PDS-1000/He are used as gene delivery systems. The herbicide resistant bar gene and selection agent bialaphos are used for identifying putative transgenic type I callus events. Using the step-by-step protocols described here, average transformation frequencies (number of bialaphos resistant T 0 callus events per 100 explants infected or bombarded) of 4% and 8% can be achieved using the Agrobacterium- and biolistic-mediated methods, respectively. An estimated duration of 16-21 weeks is needed using either protocol from the start of transformation experiments to obtaining putative transgenic plantlets with established roots. In addition to laboratory in vitro procedures, detailed greenhouse protocols for producing immature ears as transformation starting material and caring for transgenic plants for seed production are also described.

One of the major limitations of maize transformation is the isolation of a large number of immature embryos using the time-consuming manual extraction method. In this article, we describe a novel bulk embryo extraction method for fast isolation of a large number of embryos suitable for both biolistic- and Agrobacterium-mediated transformation. Optimal gene delivery and tissue culture conditions are also described for achieving high efficiency in Agrobacterium-mediated maize transformation using phosphomannose isomerase (PMI) as a selectable marker.

Agrobacterium-mediated transformation is a powerful strategy for plant genetic engineering. However, the transformation efficiency of some grain legume crops is generally low. In this chapter, we describe a chemical screening procedure for improving the efficiency of Agrobacterium-mediated transformation of a model legume, Lotus japonicus. Moreover, we explain the transformation protocol using chloroxynil, a phenolic compound that improves the efficiency.

In most bacteria, a global level of regulation, termed quorum sensing (QS), exists involving intercellular communication via the production and response to cell density-dependent signal molecules. QS has been associated with a number of important features in bacteria including virulence regulation and biofilm formation. Consequently, there is considerable interest in understanding, detecting, and inhibiting QS. N-acylated homoserine lactones (AHLs) are used as extracellular QS signals by a variety of Gram-negative bacteria. Chromobacterium violaceum, commonly found in soil and water, produces the characteristic purple pigment violacein, regulated by AHL-mediated QS. Based on this readily observed pigmentation phenotype, C. violaceum strains can be used to detect various aspects of AHL-mediated QS activity. In another commonly used bioassay organism, Agrobacterium tumefaciens, QS can be detected by the use of a reporter gene such as lacZ. Here, we describe several commonly used approaches incorporating C. violaceum and A. tumefaciens that can be used to detect AHL and QS inhibitors. Due to the inherent low susceptibility of biofilm bacteria to antimicrobial agents, biofilm dispersion, whereby bacteria reenter the planktonic community, is another increasingly important area of research. At least one signal, distinct from traditional QS, has been identified and there are a variety of other environmental factors that also trigger dispersion. We describe a microtiter-based experimental strategy whereby potential biofilm dispersion compounds can be screened.