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STAR Protocols Dec 2022We present a protocol for -mediated genetic transformation and genome editing in wild allotetraploid rice . We detail steps to induce embryogenic calli from mature seeds...
We present a protocol for -mediated genetic transformation and genome editing in wild allotetraploid rice . We detail steps to induce embryogenic calli from mature seeds and co-cultivate with after infection. We further describe how to select transformed cells that proliferated from infected calli based on hygromycin resistance, resulting in regeneration of transformed plantlets with multiple edited alleles. Edited lines produced this protocol can be used for gene functions studies and domestication of For complete details on the use and execution of this protocol, please refer to Yu et al. (2021).
Topics: Agrobacterium; Gene Editing; Oryza; Plants, Genetically Modified; Transformation, Genetic; Genome, Plant
PubMed: 36325580
DOI: 10.1016/j.xpro.2022.101789 -
ACS Chemical Biology Jan 2021Siderophores are iron-chelating molecules produced by microorganisms and plants to acquire exogenous iron. Siderophore biosynthetic enzymology often produces elaborate...
Siderophores are iron-chelating molecules produced by microorganisms and plants to acquire exogenous iron. Siderophore biosynthetic enzymology often produces elaborate and unique molecules through unusual reactions to enable specific recognition by the producing organisms. Herein, we report the structure of two siderophore analogs from strain C58, which we named fabrubactin (FBN) A and FBN B. Additionally, we characterized the substrate specificities of the NRPS and PKS components. The structures suggest unique Favorskii-like rearrangements of the molecular backbone that we propose are catalyzed by the flavin-dependent monooxygenase, FbnE. FBN A and B contain a 1,1-dimethyl-3-amino-1,2,3,4-tetrahydro-7,8-dihydroxy-quinolin (Dmaq) moiety previously seen only in the anachelin cyanobacterial siderophores. We provide evidence that Dmaq is derived from l-DOPA and propose a mechanism for the formation of the mature Dmaq moiety. Our bioinformatic analyses suggest that FBN A and B and the anachelins belong to a large and diverse siderophore family widespread throughout the / group, α-proteobacteria, and cyanobacteria.
Topics: Adenosine Monophosphate; Agrobacterium; Molecular Structure; Siderophores; Spectrum Analysis; Substrate Specificity
PubMed: 33373180
DOI: 10.1021/acschembio.0c00809 -
Communications Biology Sep 2023Large gaps exist in our understanding of how bacteriophages, the most abundant biological entities on Earth, assemble and function. The structure of the "neck" region,...
Large gaps exist in our understanding of how bacteriophages, the most abundant biological entities on Earth, assemble and function. The structure of the "neck" region, where the DNA-filled capsid is connected to the host-recognizing tail remains poorly understood. We describe cryo-EM structures of the neck, the neck-capsid and neck-tail junctions, and capsid of the Agrobacterium phage Milano. The Milano neck 1 protein connects the 12-fold symmetrical neck to a 5-fold vertex of the icosahedral capsid. Comparison of Milano neck 1 homologs leads to four proposed classes, likely evolved from the simplest one in siphophages to more complex ones in myo- and podophages. Milano neck is surrounded by the atypical collar, which covalently crosslinks the tail sheath to neck 1. The Milano capsid is decorated with three types of proteins, a minor capsid protein (mCP) and two linking proteins crosslinking the mCP to the major capsid protein. The extensive network of disulfide bonds within and between neck, collar, capsid and tail provides an exceptional structural stability to Milano.
Topics: Capsid; Capsid Proteins; Bacteriophages; Dendritic Spines; Agrobacterium
PubMed: 37684529
DOI: 10.1038/s42003-023-05292-1 -
Annual Review of Microbiology 2005Type IV secretion (T4S) systems are ancestrally related to bacterial conjugation machines. These systems assemble as a translocation channel, and often also as a surface... (Review)
Review
Type IV secretion (T4S) systems are ancestrally related to bacterial conjugation machines. These systems assemble as a translocation channel, and often also as a surface filament or protein adhesin, at the envelopes of Gram-negative and Gram-positive bacteria. These organelles mediate the transfer of DNA and protein substrates to phylogenetically diverse prokaryotic and eukaryotic target cells. Many basic features of T4S are known, including structures of machine subunits, steps of machine assembly, substrates and substrate recognition mechanisms, and cellular consequences of substrate translocation. A recent advancement also has enabled definition of the translocation route for a DNA substrate through a T4S system of a Gram-negative bacterium. This review emphasizes the dynamics of assembly and function of model conjugation systems and the Agrobacterium tumefaciens VirB/D4 T4S system. We also summarize salient features of the increasingly studied effector translocator systems of mammalian pathogens.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; Conjugation, Genetic; DNA, Bacterial; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Protein Transport; Transformation, Genetic
PubMed: 16153176
DOI: 10.1146/annurev.micro.58.030603.123630 -
BMC Plant Biology Feb 2024Crown gall disease caused by Agrobacterium tumefaciens is a very destructive affliction that affects grapevines. Endophytic bacteria have been discovered to control...
Antivirulence effects of cell-free culture supernatant of endophytic bacteria against grapevine crown gall agent, Agrobacterium tumefaciens, and induction of defense responses in plantlets via intact bacterial cells.
BACKGROUND
Crown gall disease caused by Agrobacterium tumefaciens is a very destructive affliction that affects grapevines. Endophytic bacteria have been discovered to control plant diseases via the use of several mechanisms. This research examined the potential for controlling crown gall by three endophytic bacteria that were previously isolated from healthy cultivated and wild grapevines including Pseudomonas kilonensis Ba35, Pseudomonas chlororaphis Ba47, and Serratia liquefaciens Ou55.
RESULT
At various degrees, three endophytic bacteria suppressed the populations of A. tumefaciens Gh1 and greatly decreased the symptoms of crown gall. Furthermore, biofilm production and motility behaviors of A. tumefaciens Gh1were greatly inhibited by the Cell-free Culture Supernatant (CFCS) of endophytic bacteria. According to our findings, CFCS may reduce the adhesion of A. tumefaciens Gh1 cells to grapevine cv. Rashe root tissues as well as their chemotaxis motility toward the extract of the roots. When compared to the untreated control, statistical analysis showed that CFCS significantly reduced the swimming, twitching, and swarming motility of A. tumefaciens Gh1. The findings demonstrated that the endophytic bacteria effectively stimulated the production of plant defensive enzymes including superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), and total soluble phenols at different time intervals in grapevine inoculated with A. tumefaciens Gh1. The Ba47 strain markedly increased the expression levels of defense genes associated with plant resistance. The up-regulation of PR1, PR2, VvACO1, and GAD1 genes in grapevine leaves indicates the activation of SA and JA pathways, which play a role in enhancing resistance to pathogen invasion. The results showed that treating grapevine with Ba47 increased antioxidant defense activities and defense-related gene expression, which reduced oxidative damage caused by A. tumefaciens and decreased the incidence of crown gall disease.
CONCLUSION
This is the first study on how A. tumefaciens, the grapevine crown gall agent, is affected by CFCS generated by endophytic bacteria in terms of growth and virulence features. To create safer plant disease management techniques, knowledge of the biocontrol processes mediated by CFCS during microbial interactions is crucial.
Topics: Plant Tumors; Agrobacterium tumefaciens; Plant Diseases; Bacteria
PubMed: 38336608
DOI: 10.1186/s12870-024-04779-1 -
PloS One 2020Optimizing the gene transformation factors can be considered as the first and foremost step in successful genetic engineering and genome editing studies. However, it is...
Forecasting and optimizing Agrobacterium-mediated genetic transformation via ensemble model- fruit fly optimization algorithm: A data mining approach using chrysanthemum databases.
Optimizing the gene transformation factors can be considered as the first and foremost step in successful genetic engineering and genome editing studies. However, it is usually difficult to achieve an optimized gene transformation protocol due to the cost and time-consuming as well as the complexity of this process. Therefore, it is necessary to use a novel computational approach such as machine learning models for analyzing gene transformation data. In the current study, three individual machine learning models including Multi-Layer Perceptron (MLP), Adaptive Neuro-Fuzzy Inference System (ANFIS), and Radial Basis Function (RBF) were developed for forecasting Agrobacterium-mediated gene transformation in chrysanthemum based on eleven input variables including Agrobacterium strain, optical density (OD), co-culture period (CCP), and different antibiotics including kanamycin (K), vancomycin (VA), cefotaxime (CF), hygromycin (H), carbenicillin (CA), geneticin (G), ticarcillin (TI), and paromomycin (P). Consequently, best-obtained results were used in the fusion process by bagging method. Results showed that ensemble model with the highest R2 (0.83) had superb performance in comparison with all other individual models (MLP:063, RBF:0.69, and ANFIS: 0.74) in the validation set. Also, ensemble model was linked to Fruit fly optimization algorithm (FOA) for optimizing gene transformation, and the results showed that the maximum gene transformation efficiency (37.54%) can be achieved from EHA105 strain with 0.9 OD600, for 3.8 days CCP, 46.43 mg/l P, 9.54 mg/l K, 18.62 mg/l H, and 4.79 mg/l G as selection antibiotics and 109.74 μg/ml VA, 287.63 μg/ml CF, 334.07 μg/ml CA and 87.36 μg/ml TI as antibiotics in the selection medium. Moreover, sensitivity analysis demonstrated that input variables have a different degree of importance in gene transformation system in the order of Agrobacterium strain > CCP > K > CF > VA > P > OD > CA > H > TI > G. Generally, the developed hybrid model in this study (ensemble model-FOA) can be employed as an accurate and reliable approach in future genetic engineering and genome editing studies.
Topics: Agrobacterium; Algorithms; Anti-Bacterial Agents; Bacterial Proteins; Chrysanthemum; Databases, Genetic; Genetic Engineering; Plants, Genetically Modified; Transformation, Genetic
PubMed: 32997694
DOI: 10.1371/journal.pone.0239901 -
Molecules (Basel, Switzerland) Aug 2021Vahl. is an interesting source of abietane and -abietane compounds that showed antibacterial, antitumor, and cytotoxic activities. The aim of the study was to obtain...
Vahl. is an interesting source of abietane and -abietane compounds that showed antibacterial, antitumor, and cytotoxic activities. The aim of the study was to obtain transformed roots of and to evaluate the production of terpenoids in comparison with in vivo root production. Hairy roots were initiated from leaf explants by infection with ATCC 15834 onto hormone-free Murashige and Skoog (MS) solid medium. Transformation was confirmed by polymerase chain reaction analysis of and genes. The biomass production was obtained in hormone-free liquid MS medium using Temporary Immersion System bioreactor RITA. The chromatographic separation of the methanolic extract of the untransformed roots afforded horminone, ferruginol, 7--acetylhorminone and 7--methylhorminone. Agastol and ferruginol were isolated and quantified from the hairy roots. The amount of these metabolites indicated that the hairy roots of can be considered a source of these compounds.
Topics: Abietanes; Agrobacterium; Biomass; Bioreactors; Culture Media; Diterpenes; Plant Roots; Plants, Genetically Modified; Salvia; Transformation, Genetic
PubMed: 34500582
DOI: 10.3390/molecules26175144 -
BMC Microbiology Oct 2021The Agrobacterium strain AB2/73 has a unique host range for the induction of crown gall tumors, and contains an exceptionally large, over 500 kbp mega Ti plasmid. We...
BACKGROUND
The Agrobacterium strain AB2/73 has a unique host range for the induction of crown gall tumors, and contains an exceptionally large, over 500 kbp mega Ti plasmid. We used whole genome sequencing to fully characterize and comparatively analyze the complex genome of strain AB2/73, including its Ti plasmid and virulence factors.
RESULTS
We obtained a high-quality, full genomic sequence of AB2/73 by a combination of short-read Illumina sequencing and long-read Nanopore sequencing. The AB2/73 genome has a total size of 7,266,754 bp with 59.5% GC for which 7012 genes (6948 protein coding sequences) are predicted. Phylogenetic and comparative genomics analysis revealed that strain AB2/73 does not belong to the genus Agrobacterium, but to a new species in the genus Rhizobium, which is most related to Rhizobium tropici. In addition to the chromosome, the genome consists of 6 plasmids of which the largest two, of more than 1 Mbp, have chromid-like properties. The mega Ti plasmid is 605 kbp in size and contains two, one of which is incomplete, repABC replication units and thus appears to be a cointegrate consisting of about 175 kbp derived from an unknown Ti plasmid linked to 430 kbp from another large plasmid. In pTiAB2/73 we identified a complete set of virulence genes and two T-DNAs. Besides the previously described T-DNA we found a larger, second T-DNA containing a 6b-like onc gene and the acs gene for agrocinopine synthase. Also we identified two clusters of genes responsible for opine catabolism, including an acc-operon for agrocinopine degradation, and genes putatively involved in ridéopine catabolism. The plasmid also harbours tzs, iaaM and iaaH genes for the biosynthesis of the plant growth regulators cytokinin and auxin.
CONCLUSIONS
The comparative genomics analysis of the high quality genome of strain AB2/73 provided insight into the unusual phylogeny and genetic composition of the limited host range Agrobacterium strain AB2/73. The description of its unique genomic composition and of all the virulence determinants in pTiAB2/73 will be an invaluable tool for further studies into the special host range properties of this bacterium.
Topics: Agrobacterium; DNA, Bacterial; Genes, Bacterial; Genome, Bacterial; Genomics; Host Specificity; Phylogeny; Plant Tumors; Plasmids; Replicon; Rhizobium; Virulence
PubMed: 34711172
DOI: 10.1186/s12866-021-02358-0 -
MBio Oct 2021The type VI secretion system (T6SS) is used by many Gram-negative bacteria to deploy toxic effectors for interbacterial competition. This system provides a competitive...
The type VI secretion system (T6SS) is used by many Gram-negative bacteria to deploy toxic effectors for interbacterial competition. This system provides a competitive advantage to agrobacteria, a diverse group with phytopathogenic members capable of genetically transforming plants. To inform on the ecology and evolution of agrobacteria, we revealed processes that diversify their effector gene collections. From genome sequences of diverse strains, we identified T6SS loci, functionally validated associated effector genes for toxicity, and predicted genes homologous to those that encode proteins known to interact with effectors. The gene loci were analyzed in a phylogenetic framework, and results show that strains of some species-level groups have different patterns of T6SS expression and are enriched in specific sets of T6SS loci. Findings also demonstrate that the modularity of T6SS loci and their associated genes engenders dynamicity, promoting reshuffling of entire loci, fragments therein, and domains to swap toxic effector genes across species. However, diversification is constrained by the need to maintain specific combinations of gene subtypes, congruent with observations that certain genes function together to regulate T6SS loading and activation. Data are consistent with a scenario where species can acquire unique T6SS loci that are then reshuffled across the genus in a restricted manner to generate new combinations of effector genes. The T6SS is used by several taxa of Gram-negative bacteria to secrete toxic effector proteins to attack others. Diversification of effector collections shapes bacterial interactions and impacts the health of hosts and ecosystems in which bacteria reside. We uncovered the diversity of T6SS loci across a genus of plant-associated bacteria and show that diversification is driven by the acquisition of new loci and reshuffling among species. However, linkages between specific subtypes of genes need to be maintained to ensure that proteins whose interactions are necessary to activate the T6SS remain together. Results reveal how organization of gene loci and domain structure of genes provides flexibility to diversify under the constraints imposed by the system. Findings inform on the evolution of a mechanism that influences bacterial communities.
Topics: Agrobacterium; Bacterial Proteins; Bacterial Secretion Systems; Ecosystem; Evolution, Molecular; Phylogeny; Type VI Secretion Systems
PubMed: 34517758
DOI: 10.1128/mBio.01927-21 -
Annual Review of Phytopathology Aug 2019Genetic transformation of host plants by and related species represents a unique model for natural horizontal gene transfer. Almost five decades of studying the...
Genetic transformation of host plants by and related species represents a unique model for natural horizontal gene transfer. Almost five decades of studying the molecular interactions between and its host cells have yielded countless fundamental insights into bacterial and plant biology, even though several steps of the DNA transfer process remain poorly understood. spp. may utilize different pathways for transferring DNA, which likely reflects the very wide host range of . Furthermore, closely related bacterial species, such as rhizobia, are able to transfer DNA to host plant cells when they are provided with DNA transfer machinery and T-DNA. Homologs of virulence genes are found in many bacterial genomes, but only one non- bacterial strain, CFN42, harbors a complete set of virulence genes and can mediate plant genetic transformation when carrying a T-DNA-containing plasmid.
Topics: Agrobacterium tumefaciens; Bacteria; Bacterial Proteins; DNA, Bacterial; Gene Transfer, Horizontal; Plants; Virulence
PubMed: 31226020
DOI: 10.1146/annurev-phyto-082718-100101