Tumefaciens Strain C58 Research Articles

Promoter Prediction in Agrobacterium tumefaciens Strain C58 by Using Artificial Intelligence Strategies.

Promoters are the genomic regions upstream of genes that RNA polymerase binds in order to initiate gene transcription. Understanding the regulation of gene expression depends on being able to identify promoters, because they are the most important component of gene expression. Agrobacterium tumefaciens (A. tumefaciens) strain C58 was the subject of this study with the goal of creating a machine learning-based model to predict promoters. In this study, nucleotide density (ND), k-mer, and one-hot were used to encode the promoter sequence. Support vector machine (SVM) on fivefold cross-validation with incremental feature selection (IFS) was used to optimize the generated features. These improved characteristics were then used to distinguish promoter sequences by feeding them into the random forest (RF) classifier. Tenfold cross-validation (CV) analysis revealed that the projected model has the ability to produce an accuracy of 84.22%.

Computational prediction of promotors in Agrobacterium tumefaciens strain C58 by using the machine learning technique.

Promotors are those genomic regions on the upstream of genes, which are bound by RNA polymerase for starting gene transcription. Because it is the most critical element of gene expression, the recognition of promoters is crucial to understand the regulation of gene expression. This study aimed to develop a machine learning-based model to predict promotors in Agrobacterium tumefaciens (A. tumefaciens) strain C58. In the model, promotor sequences were encoded by three different kinds of feature descriptors, namely, accumulated nucleotide frequency, k-mer nucleotide composition, and binary encodings. The obtained features were optimized by using correlation and the mRMR-based algorithm. These optimized features were inputted into a random forest (RF) classifier to discriminate promotor sequences from non-promotor sequences in A. tumefaciens strain C58. The examination of 10-fold cross-validation showed that the proposed model could yield an overall accuracy of 0.837. This model will provide help for the study of promoters in A. tumefaciens C58 strain.

Transformation of Kalanchoe pinnata by Agrobacterium tumefaciens with ZsGreen1

Screening tests in which 30 species of succulent plants were inoculated with A136 or C58 strains of Agrobacterium tumefaciens led to selecting genus Kalanchoe for further study of Agrobacterium-mediated transformation. Regeneration studies showed that 4.55 µM Thidiazuron (TDZ) combined with 2.15 µM α-naphtaleneacetic acid (NAA) significantly proliferated callus development from Kalanchoe pinnata leaf explants in Murashige and Skoog (MS) medium. Subsequently, 4.55 µM TDZ significantly enhanced shoot formation from the green and compact callus. NAA (5.37 µM) accelerated root growth leading to establishment of transgenic plants in soil. A gene encoding the fluorescent protein, ZsGreen1, was cloned into a plasmid vector harboring a constitutive figwort mosaic virus (FMV) 35S promoter, a kanamycin resistance gene, and a NOS-terminator. The plasmids were delivered into the genome of K. pinnata by A. tumefaciens strain C58 (ABI). The ZsGreen1-transgenic plants exhibited very strong, green fluorescence from the callus, leaves, stems, roots, and flowers visible through a green emission filter (GFP, 480/510 nm, excitation/emission) under excitation by ultra-violet light. Transcription analysis by sqRT-PCR and qPCR confirmed the stable integration of the ZsGreen1 in the genome of K. pinnata. The FMV 35S promoter conferred uniform expression throughout whole plant organ. ZsGreen1 was proven to be a stable marker for gene expression in genetic transformation of K. pinnata. The ZsGreen1-transgenic Kalanchoe pinnata mediated by Agrobacterium tumefaciens exhibited strong, green fluorescence from the callus, leaves, stems, roots and flowers under the control of the figwort mosaic virus (FMV) 35S promoter.

A High-Throughput Interbacterial Competition Screen Identifies ClpAP in Enhancing Recipient Susceptibility to Type VI Secretion System-Mediated Attack by Agrobacterium tumefaciens.

The type VI secretion system (T6SS) is an effector delivery system used by Gram-negative bacteria to kill other bacteria or eukaryotic hosts to gain fitness. The plant pathogen Agrobacterium tumefaciens utilizes its T6SS to kill other bacteria, such as Escherichia coli. We observed that the A. tumefaciens T6SS-dependent killing outcome differs when using different T6SS-lacking, K-12 E. coli strains as a recipient cell. Thus, we hypothesized that the A. tumefaciens T6SS killing outcome not only relies on the T6SS activity of the attacker cells but also depends on the recipient cells. Here, we developed a high-throughput interbacterial competition platform to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. Among the 3,909 strains in the E. coli Keio library screened, 16 mutants with less susceptibility to A. tumefaciens C58 T6SS-dependent killing were identified, and four of them were validated by complementation test. Among the four, the clpP encoding ClpP protease, which is universal and highly conserved in both prokaryotes and eukaryotic organelles, was selected for further characterizations. We demonstrated that ClpP is responsible for enhancing susceptibility to the T6SS killing. Because ClpP protease depends on other adapter proteins such as ClpA and ClpX for substrate recognition, further mutant studies followed by complementation tests were carried out to reveal that ClpP-associated AAA+ ATPase ClpA, but not ClpX, is involved in enhancing susceptibility to A. tumefaciens T6SS killing. Moreover, functional and biochemical studies of various ClpP amino acid substitution variants provided evidence that ClpA–ClpP interaction is critical in enhancing susceptibility to the T6SS killing. This study highlights the importance of recipient factors in determining the outcome of the T6SS killing and shows the universal ClpP protease as a novel recipient factor hijacked by the T6SS of A. tumefaciens.

Effector loading onto the VgrG carrier activates type VI secretion system assembly

The type VI secretion system (T6SS) is used by many bacteria to engage in social behavior and can affect the health of its host plant or animal. Because activities associated with T6SSs are often costly, T6SSs must be tightly regulated. However, our knowledge regarding how T6SS assembly and contraction are regulated remains limited. Using the plant pathogen Agrobacterium tumefaciens, we show that effectors are not just passengers but also impact on T6SS assembly. The A. tumefaciens strain C58 encodes one T6SS and two Tde DNase toxin effectors used as major weapons for interbacterial competition. Here, we demonstrate that loading of Tde effectors onto their cognate carriers, the VgrG spikes, is required for active T6SS secretion. The assembly of the TssBC contractile sheath occurs only in the presence of Tde effectors. The requirement of effector loading for efficient T6SS secretion was also validated in other A. tumefaciens strains. We propose that such a mechanism is used by bacteria as a strategy for efficacious T6SS firing and to ensure that effectors are loaded onto the T6SS prior to completing its assembly.

Overexpression of the ZmDEF1 gene increases the resistance to weevil larvae in transgenic maize seeds.

Plant defensins are divided into 18 groups and are multifunctional proteins. The Zea mays defensin 1 (ZmDEF1) gene encodes the defensin 1 protein, which can inhibit alpha-amylase in the insect gut. In this study, the ZmDEF1 gene was transferred into two maize cultivars, LC1 and LVN99, to improve weevil resistance in maize. The recombinant ZmDEF1 protein was assessed for its ability to inhibit alpha-amylase in the gut of the larvae of the maize weevil (Sitophilus zeamais Motsch.). ZmDEF1 was cloned into a pBetaPhaso-dest vector, which harbours phaseolin, a seed-specific promoter, and the Agrobacterium tumefaciens strain C58 harbouring the pBetaPhaso-ZmDEF1 vector was used to transfer the ZmDEF1 gene into two maize cultivars using immature embryos. Transformed calluses were selected on selection media containing kanamycin. The stable integration of the ZmDEF1 transgene into the transgenic maize plant genome was confirmed using Southern blotting. The recombinant ZmDEF1 protein of approximately 10kDa was expressed in three transgenic maize lines from the LC1 cultivar (C1, C3, and C5) and two transgenic maize lines from the LVN99 cultivar (L1 and L3). The ZmDEF1 transgenic efficiency based on the results of PCR, as well as Southern and Western blotting, was 1.32% and 0.82%, respectively, which depends on the genotypes of LC1 and LVN99. The recombinant ZmDEF1 protein inhibited the alpha-amylase activity of the maize weevil larvae, and its ability to inhibit alpha-amylase is 54.52-63.09% greater than the ZmDEF1 protein extracted from non-transgenic plants.

Lipopeptides from Bacillus amyloliquefaciens strain 32a as promising biocontrol compounds against the plant pathogen Agrobacterium tumefaciens.

Despite the potential biological importance of lipopeptides from Bacillus amyloliquefaciens as antimicrobial compounds, their effects on Agrobacterium tumefaciens biofilms have not been previously studied. These latter are important virulence factors for the development and re-occurrence of crown gall disease. As part of the development of a new biopesticide acting as anti-biofilm and biocontrol agent, we investigated for the first time the ability of a mixture of lipopeptides produced by B. amyloliquefaciens strain 32a to inhibit the tumor formation on plants and to reduce the formation of biofilms by the phytopathogenic A. tumefaciens strains C58 and B6. The mixture was found to display a strong biosurfactant activity as well as bactericidal activity against planktonic Agrobacterium cells. Moreover, the lipopeptide treatment inhibited biofilm formation in the range of 79.58 ± 0.60-100.00 ± 0.00% and dislodged 43.42 ± 0.91-93.89 ± 2.70% of preformed biofilm. For these assays, fluorescence microscopy did not show any adherent cell in the anti-adhesive assay and only few ones in the cell-dislodging assay. More importantly, lipopeptide-enriched extract inhibits tumor formation on tomato stem when treatments were applied after pathogen adhesion to wounded tissues. By virtue of its ability to inhibit biofilms formed on biotic and abiotic surfaces and to control efficiently tumor development, the 32a lipopeptide mixture may represent an excellent new tool for an efficient biocontrol of crown gall disease.

Larger Than Life: Isolation and Genomic Characterization of a Jumbo Phage That Infects the Bacterial Plant Pathogen, Agrobacterium tumefaciens

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease, leading to the damage of agriculturally-important crops. As part of an effort to discover new phages that can potentially be used as biocontrol agents to prevent crown gall disease, we isolated and characterized phage Atu_ph07 from Sawyer Creek in Springfield, MO, using the virulent Agrobacterium tumefaciens strain C58 as a host. After surveying its host range, we found that Atu_ph07 exclusively infects Agrobacterium tumefaciens. Time-lapse microscopy of A. tumefaciens cells subjected to infection at a multiplicity of infection (MOI) of 10 with Atu_ph07 reveals that lysis occurs within 3 h. Transmission electron microscopy (TEM) of virions shows that Atu_ph07 has a typical Myoviridae morphology with an icosahedral head, long tail, and tail fibers. The sequenced genome of Atu_ph07 is 490 kbp, defining it as a jumbo phage. The Atu_ph07 genome contains 714 open reading frames (ORFs), including 390 ORFs with no discernable homologs in other lineages (ORFans), 214 predicted conserved hypothetical proteins with no assigned function, and 110 predicted proteins with a functional annotation based on similarity to conserved proteins. The proteins with predicted functional annotations share sequence similarity with proteins from bacteriophages and bacteria. The functionally annotated genes are predicted to encode DNA replication proteins, structural proteins, lysis proteins, proteins involved in nucleotide metabolism, and tRNAs. Characterization of the gene products reveals that Atu_ph07 encodes homologs of 16 T4 core proteins and is closely related to Rak2-like phages. Using ESI-MS/MS, the majority of predicted structural proteins could be experimentally confirmed and 112 additional virion-associated proteins were identified. The genomic characterization of Atu_ph07 suggests that this phage is lytic and the dynamics of Atu_ph07 interaction with its host indicate that this phage may be suitable for inclusion in a phage cocktail to be used as a biocontrol agent.

Cloning and constructing transformation vector carrying IbOr from the sweetpotato (Impomea batatas L.) involved in accumulation of carotenoids

The Orange gene (Or gene) is known as a gene encoding the protein containing a cysteine-rich Zinc finger domain. The Or protein is an important component of the structure that involes in accumulation of carotenoids and enhancing the resistant ability of plant to enviromental stresses. In an attempt to improve the nutritional quality and resistance to adverse environmental conditions of maize, in this article, we present the results on cloning IbOr gene from the sweetpotato cultivar Hoang Long and constructing transformation vectors carrying cloned IbOr gene under control of Ubiquitin (Ubi) or Globulin 1 (Glo1) promoter. The cloned IbOr gene was 942 bp in size and had similarity level of 100% comparing to the IbOr gene that was deposited in GenBank with Accession number HQ828087.1. The IbOr gene from sweedpotato Hoang Long was registered in GenBank under Accession number KX792094.1. The deduced protein consisted of 313 amino acids having Zin finger domain of DnaJ and HSP40 proteins. We have also constructed two transformation vectors pCambia2300/Ubiquitin/IbOr/Nos and pCambia2300/Glo1/IbOr/Nos. These vectors were transformed successfully into Agrobacterium tumefaciens strain C58. The presence of the target gene (IbOr) and promoter (Ubi or Glo1) in the A. tumefaciens strains were confirmed by colony-PCR amplification with respective specific primer pairs and digested by suitable restriction enzymes. These vectors will provide important materials for further gene transfer research for the expression of IbOr gene and production of transgenic maize with ability to accumulate high carotenoids.

Production of transgenic tobacco plants expressing GS1 gene for the increase ofnitrogen use efficiency

Glutamine synthetase (GS, CE 6.3.1.2) is an enzyme that catalyzes the ATP-dependent condensation of glutamic acid with ammonia to yield glutamine. Glutamine synthetase is a key enzyme involved in the assimilation of inorganic nitrogen into organic forms. In plant cells, GS is present in both chloroplasts (GS2) and cytoplasm (GS1), in which GS1 can assimilate nitrogen source. In this study, one transgenic vector pBI121 carrying GS1 gene under the control of promoter 35S (pBI121::GS1) were successfully constructed. This vector containing G1S gene was transformed into tobacco leaves pieces via Agrobacterium tumefaciens strain C58. Five weeks after cultivation, there were 28 tobacco lines which had roots on the medium added kanamycin 50 mg/l. Then, the presence of G1S gene in these tobacco lines was tested from leaves in the next experiments. PCR and Southern blot confirmed that there are five tobacco lines carrying transferred GS1 gene. The effectiveness of nitrogen using in GS1 transgenic tobacco plants in vitro was evaluated. The tissue fresh weight, number and height of shoots forming buds and rooting ability of GS1 transgenic tobacco plants were greater than those of non-GM plants in the medium of low nitrogen concentration (0.1X - 0.2X). Assessment of crop growing in a greenhouse demonstrated that GS1 transgenic tobacco plants grow faster than non-transgenic ones. In detail, the increment of plant height after planting 03 months and 05 months in greenhouse is 43.55% and 33.29%, respectively. These results provide a scientific basis for the development of other genetically modified plants which enhanced nitrogen-use efficiency.

The Nonspecific Lipid Transfer Protein AtLtpI-4 Is Involved in Suberin Formation of Arabidopsis thaliana Crown Galls.

Nonspecific lipid transfer proteins reversibly bind different types of lipid molecules in a hydrophobic cavity. They facilitate phospholipid transfer between membranes in vitro, play a role in cuticle and possibly in suberin formation, and might be involved in plant pathogen defense signaling. This study focuses on the role of the lipid transfer protein AtLTPI-4 in crown gall development. Arabidopsis (Arabidopsis thaliana) crown gall tumors, which develop upon infection with the virulent Agrobacterium tumefaciens strain C58, highly expressed AtLTPI-4 Crown galls of the atltpI-4 loss-of-function mutant were much smaller compared with those of wild-type plants. The gene expression pattern and localization of the protein to the plasma membrane pointed to a function of AtLTPI-4 in cell wall suberization. Since Arabidopsis crown galls are covered by a suberin-containing periderm instead of a cuticle, we analyzed the suberin composition of crown galls and found a reduction in the amounts of long-chain fatty acids (C18:0) in the atltpI-4 mutant. To demonstrate the impact of AtLtpI-4 on extracellular lipid composition, we expressed the protein in Arabidopsis epidermis cells. This led to a significant increase in the very-long-chain fatty acids C24 and C26 in the cuticular wax fraction. Homology modeling and lipid-protein-overlay assays showed that AtLtpI-4 protein can bind these very-long-chain fatty acids. Thus, AtLtpI-4 protein may facilitate the transfer of long-chain as well as very-long-chain fatty acids into the apoplast, depending on the cell type in which it is expressed. In crown galls, which endogenously express AtLtpI-4, it is involved in suberin formation.

Artificial Agrobacterium tumefaciens strains exhibit diverse mechanisms to repress Xanthomonas oryzae pv. oryzae-induced hypersensitive response and non-host resistance in Nicotiana benthamiana.

Xanthomonas oryzae pv. oryzae (Xoo) rapidly triggers a hypersensitive response (HR) and non-host resistance in its non-host plant Nicotiana benthamiana. Here, we report that Agrobacterium tumefaciens strain GV3101 blocks Xoo-induced HR in N.benthamiana when pre-infiltrated or co-infiltrated, but not when post-infiltrated at 4h after Xoo inoculation. This suppression by A.tumefaciens is local and highly efficient to Xoo. The HR-inhibiting efficiency of A.tumefaciens is strain dependent. Strain C58C1 has almost no effect on Xoo-induced HR, whereas strains GV3101, EHA105 and LBA4404 nearly completely block HR formation. Intriguingly, these three HR-inhibiting strains employ different strategies to repress HR. Strain GV3101 displays strong antibiotic activity and thus suppresses Xoo growth. Comparison of the genotype and Xoo antibiosis activity of wild-type A.tumefaciens strain C58 and a set of C58-derived strains reveals that this Xoo antibiosis activity of A.tumefaciens is negatively, but not solely, regulated by the transferred-DNA (T-DNA) of the Ti plasmid pTiC58. Unlike GV3101, strains LBA4404 and EHA105 exhibit no significant antibiotic effect on Xoo, but rather abolish hydrogen peroxide accumulation. In addition, expression assays indicate that strains LBA4404 and EHA105 may inhibit Xoo-induced HR by suppression of the expression of Xoo type III secretion system (T3SS) effector genes hpa1 and hrpD6. Collectively, our results unveil the multiple levels of effects of A.tumefaciens on Xoo in N.benthamiana and provide insights into the molecular mechanisms underlying the bacterial antibiosis of A.tumefaciens and the non-host resistance induced by Xoo.

Agrobacterium-mediated transformation of five inbred maize lines with the Brittle 2 gene

The Brittle 2 (Bt2) gene encodes a small subunit of ADP-glucose pyrophosphorylase (AGPase) enzyme, one of the enzymes that play an important role in endosperm starch biosynthesis. We carried out a study to transfer the Bt2 gene into several inbred maize lines and regenerate transgenic maize plants possessing the transgene, which can be used to further study the role of the Bt2 gene in starch biosynthesis. Agrobacterium tumefaciens strains C58 and EHA105 harboring the transformation vector pCambia2300/Ubi-Bt2-Nos were used to transfer the Bt2 gene into five inbred maize lines, H14, H26, H240, H95, and N18. Transformed calli were selected on selection medium containing 100 mg/L of kanamycin. The stable integration of the Bt2 gene into transgenic plants was confirmed by Southern hybridizations. We recorded the different transformation frequencies in the experiments with A. Tumefaciens strains C58 and EHA105. Transgenic plants were regenerated from transformed calli of all five studied maize lines. Transformation frequencies varied from 1.09% to 2.33%, depending on maize genotypes. The results obtained show that our transformation system is effective for numerous maize genotypes. H95 and H240 are good lines for gene transfer in maize.

Computational prediction of over-annotated protein-coding genes in the genome of Agrobacterium tumefaciens strain C58**Project supported by the National Natural Science Foundation of China (Grant Nos. 61302186 and 61271378) and the Funding from the State Key Laboratory of Bioelectronics of Southeast University.

Agrobacterium tumefaciens strain C58 is a type of pathogen that can cause tumors in some dicotyledonous plants. Ever since the genome of A. tumefaciens strain C58 was sequenced, the quality of annotation of its protein-coding genes has been queried continually, because the annotation varies greatly among different databases. In this paper, the questionable hypothetical genes were re-predicted by integrating the TN curve and Z curve methods. As a result, 30 genes originally annotated as “hypothetical” were discriminated as being non-coding sequences. By testing the re-prediction program 10 times on data sets composed of the function-known genes, the mean accuracy of 99.99% and mean Matthews correlation coefficient value of 0.9999 were obtained. Further sequence analysis and COG analysis showed that the re-annotation results were very reliable. This work can provide an efficient tool and data resources for future studies of A. tumefaciens strain C58.

A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3’-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

Structure and function of a decarboxylating Agrobacterium tumefaciens keto-deoxy-d-galactarate dehydratase.

Agrobacterium tumefaciens (At) strain C58 contains an oxidative enzyme pathway that can function on both d-glucuronic and d-galacturonic acid. The corresponding gene coding for At keto-deoxy-d-galactarate (KDG) dehydratase is located in the same gene cluster as those coding for uronate dehydrogenase (At Udh) and galactarolactone cycloisomerase (At Gci) which we have previously characterized. Here, we present the kinetic characterization and crystal structure of At KDG dehydratase, which catalyzes the next step, the decarboxylating hydrolyase reaction of KDG to produce α-ketoglutaric semialdehyde (α-KGSA) and carbon dioxide. The crystal structures of At KDG dehydratase and its complexes with pyruvate and 2-oxoadipic acid, two substrate analogues, were determined to 1.7 Å, 1.5 Å, and 2.1 Å resolution, respectively. Furthermore, mass spectrometry was used to confirm reaction end-products. The results lead us to propose a structure-based mechanism for At KDG dehydratase, suggesting that while the enzyme belongs to the Class I aldolase protein family, it does not follow a typical retro-aldol condensation mechanism.

Transfering Shrunken 2 (Sh2) gene coding for adp-glucose pyrophosphorylase enzyme into some inbred maize lines by Agrobacterium tumefaciens mediated gene transfer

The Shrunken 2 (Sh2) gene was proved to be involved in starch biosynthesis as it codes for two large subunits of ADP-glucose pyrophosphorylase - an enzyme that plays an important role in regulating starch biosynthesis in cereal crops. The aim of this study was to transfer Sh2 gene into some inbred maize lines and regenerate transgenic maize plants possessing transgene for further study of the role of Sh2 gene in the enhancement of starch biosynthesis in maize. The Agrobacterium tumefaciens strains C58 and DHA105 harboring transformation vector pCambia1301/Ubi/Sh2 containing Sh2 gene and monocot-specific Ubiquitin promoter were used to transfer Sh2 gene into immature embryos of the maize lines. PCR amplification of Ubi and Sh2 genes was performed to confirm the presence of the transgene in T0 transgenic plants. While, Southern and Northern hybridizations were carried out to confirm stable integration and expression of Sh2 gene in T0 and T1 transgenic plants, respectively. Transgenic plants were regenerated from transformed calli of six maize lines H95, H240, H26, H14, H4 and CML161 with the transformation frequencies from 1.08 to 1.77%. A. tumefaciens strain DHA105 gave higher transformation frequencies than that of C58 strain and the values ranged from 1.60 to 4.31%. The transgenic maize plants possessed 1 to 3 copies of transgene. Many transgenic plants grew normally and set seeds. The study of the effect of transgene in starch biosynthesis in transgenic maize lines will be performed.

The discovery of a lactone isomerase involved in pectin degradation (LB137)

Pectin, an abundant plant cell wall polymer, is being investigated as a possible raw material for the production of fuel and chemical products. To this end, an understanding of the steps to degrade pectin is useful. Agrobacterium tumefaciens strain C58 can use the primary constituent monomer of pectin, D‐galacturonate, as a sole carbon source and converts it to α‐ketoglutarate through a five step pathway in which the first step is oxidation of D‐galacturonate to D‐galactaro‐1,5‐lactone. We became interested in this pathway because a member of the functionally diverse enolase superfamily, designated Gci, catalyzes the third step in the pathway, and our lab has studied the enolase superfamily for over 20 years. We have identified an enzyme, D‐galactarolactone isomerase (GLI), adjacent to Gci in Agrobacterium’s genome, that catalyzes the second step in the pathway: the isomerization of D‐galactaro‐1,5‐lactone to D‐galactaro‐1,4‐lactone. The uncatalyzed isomerization reaction is “fast” and had been presumed to occur without the need of a catalyst. To characterize the enzyme‐catalyzed isomerization, we used 1H NMR to identify the activity, polarimetry to determine the kinetic constants (kcat = 440 s‐1, kcat/Km = 8.3 x 104 M‐1 s‐1, rate enhancement = 6.8 x 105), x‐ray crystallography to determine its structure, and gene expression analysis to connect the in vitro activity of the enzyme to an in vivo function. GLI is a member of the functionally diverse amidohydrolase superfamily and a homologue of LigI that catalyzes the hydrolysis of 2‐pyrone‐4,6‐dicarboxylate in lignin degradation. GLI’s ability to catalyze lactone isomerization instead of hydrolysis can be explained by the absence of the general basic catalysis used by LigI. This work identified the first example of lactone isomerization in the amidohydrolase superfamily and a missing enzyme in Agrobacterium’s pathway to catabolize pectin.Grant Funding Source: Supported by National Institutes of Health Grants P01GM071790 and U54GM093342 to J.A.G.

Galactaro δ-Lactone Isomerase: Lactone Isomerization by a Member of the Amidohydrolase Superfamily

Agrobacterium tumefaciens strain C58 can utilize d-galacturonate as a sole source of carbon via a pathway in which the first step is oxidation of d-galacturonate to d-galactaro-1,5-lactone. We have identified a novel enzyme, d-galactarolactone isomerase (GLI), that catalyzes the isomerizaton of d-galactaro-1,5-lactone to d-galactaro-1,4-lactone. GLI, a member of the functionally diverse amidohydrolase superfamily, is a homologue of LigI that catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate in lignin degradation. The ability of GLI to catalyze lactone isomerization instead of hydrolysis can be explained by the absence of the general basic catalysis used by 2-pyrone-4,6-dicarboxylate lactonase.

Concerted transfer of the virulence Ti plasmid and companion At plasmid in the Agrobacterium tumefaciens‐induced plant tumour

The plant pathogen Agrobacterium tumefaciens C58 harbours three independent type IV secretion (T4SS) machineries. T4SST-DNA promotes the transfer of the T-DNA to host plant cells, provoking tumour development and accumulation of opines such as nopaline and agrocinopines. T4SSpTi and T4SSpAt control the bacterial conjugation of the Ti and At plasmids respectively. Expression of T4SSpTi is controlled by the agrocinopine-responsive transcriptional repressor AccR. In this work, we compared the genome-wide transcriptional profile of the wild-type A. tumefaciens strain C58 with that of its accR KO-mutant to delineate the AccR regulon. In addition to the genes that encode agrocinopine catabolism and T4SSpTi , we found that AccR also regulated genes coding for nopaline catabolism and T4SSpAt . Further opine detection and conjugation assays confirmed the enhancement of nopaline consumption and At plasmid conjugation frequency in accR. Moreover, co-regulation of the T4SSpTi and T4SSpAt correlated with the co-transfer of the At and Ti plasmids both in vitro and in plant tumours. Finally, unlike T4SSpTi , T4SSpAt activation does not require quorum-sensing. Overall this study highlights the regulatory interplays between opines, At and Ti plasmids that contribute to a concerted dissemination of the two replicons in bacterial populations colonizing the plant tumour.