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The Plant Cell Jul 2016Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and... (Review)
Review
Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized.
Topics: Agrobacterium tumefaciens; Crops, Agricultural; DNA, Plant; Gene Editing; Genome, Plant; Recombination, Genetic; Transformation, Genetic
PubMed: 27335450
DOI: 10.1105/tpc.16.00196 -
Journal of Experimental Botany Jun 2023CRISPR/Cas9 genome editing and Agrobacterium tumefaciens-mediated genetic transformation are widely-used plant biotechnology tools derived from bacterial...
CRISPR/Cas9 genome editing and Agrobacterium tumefaciens-mediated genetic transformation are widely-used plant biotechnology tools derived from bacterial immunity-related systems, each involving DNA modification. The Cas9 endonuclease introduces DNA double-strand breaks (DSBs), and the A. tumefaciens T-DNA is released by the VirD2 endonuclease assisted by VirDl and attached by VirE2, transferred to the plant nucleus and integrated into the genome. Here, we explored the potential for synergy between the two systems and found that Cas9 and three virulence (Vir) proteins achieve precise genome editing via the homology directed repair (HDR) pathway in tobacco and rice plants. Compared with Cas9T (Cas9, VirD1, VirE2) and CvD (Cas9-VirD2) systems, the HDR frequencies of a foreign GFPm gene in the CvDT system (Cas9-VirD2, VirD1, VirE2) increased 52-fold and 22-fold, respectively. Further optimization of the CvDT process with a donor linker (CvDTL) achieved a remarkable increase in the efficiency of HDR-mediated genome editing. Additionally, the HDR efficiency of the three rice endogenous genes ACETOLACTATE SYNTHASE (ALS), PHYTOENE DESATURASE (PDS), and NITROGEN TRANSPORTER 1.1 B (NRT1.1B) increased 24-, 32- and 16-fold, respectively, in the CvDTL system, compared with corresponding Cas9TL (Cas9T process with a donor linker). Our results suggest that collaboration between CRISPR/Cas9 and Agrobacterium-mediated genetic transformation can make great progress towards highly efficient and precise genome editing via the HDR pathway.
Topics: Gene Editing; CRISPR-Cas Systems; Agrobacterium tumefaciens; Virulence; DNA
PubMed: 36919203
DOI: 10.1093/jxb/erad096 -
The New Phytologist Mar 2023Agrobacterium tumefaciens microbe-associated molecular pattern elongation factor Tu (EF-Tu) is perceived by orthologs of the Arabidopsis immune receptor EFR activating...
Agrobacterium tumefaciens microbe-associated molecular pattern elongation factor Tu (EF-Tu) is perceived by orthologs of the Arabidopsis immune receptor EFR activating pattern-triggered immunity (PTI) that causes reduced T-DNA-mediated transient expression. We altered EF-Tu in A. tumefaciens to reduce PTI and improved transformation efficiency. A robust computational pipeline was established to detect EF-Tu protein variation in a large set of plant bacterial species and identified EF-Tu variants from bacterial pathogen Pseudomonas syringae pv. tomato DC3000 that allow the pathogen to escape EFR perception. Agrobacterium tumefaciens strains were engineered to substitute EF-Tu with DC3000 variants and examined their transformation efficiency in plants. Elongation factor Tu variants with rarely occurred amino acid residues were identified within DC3000 EF-Tu that mitigates recognition by EFR. Agrobacterium tumefaciens strains were engineered by expressing DC3000 EF-Tu instead of native agrobacterial EF-Tu and resulted in decreased plant immunity detection. These engineered A. tumefaciens strains displayed an increased efficiency in transient expression in both Arabidopsis thaliana and Camelina sativa. The results support the potential application of these strains as improved vehicles to introduce transgenic alleles into members of the Brassicaceae family.
Topics: Agrobacterium tumefaciens; Arabidopsis; Arabidopsis Proteins; Gene Expression Regulation, Plant; Gene Transfer Techniques; Peptide Elongation Factor Tu; Plant Immunity; Pseudomonas syringae
PubMed: 36564969
DOI: 10.1111/nph.18694 -
Applied and Environmental Microbiology Apr 2020Acidocalcisomes are membrane-enclosed, polyphosphate-containing acidic organelles in lower but have also been described for (M. Seufferheld, M. Vieira, A. Ruiz, C. O....
Acidocalcisomes are membrane-enclosed, polyphosphate-containing acidic organelles in lower but have also been described for (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 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 with deletions of both genes () were unable to form polyphosphate but still showed cell pole-located eYFP-HppA foci and could be stained with MitoTracker. In conclusion, forms polyP granules that are free of a surrounding membrane and thus resemble polyP granules of and other bacteria. The composition, contents, and function of the subcellular structures that are stainable with MitoTracker and harbor eYFP-HppA remain unclear. 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 as an example.
Topics: Agrobacterium tumefaciens; Microscopy, Fluorescence; Organelles; Polyphosphates
PubMed: 32060025
DOI: 10.1128/AEM.02759-19 -
Microbial Physiology 2022Agrobacterium tumefaciens has two polyphosphate (polyP) kinases, one of which (PPK1AT) is responsible for the formation of polyP granules, while the other (PPK2AT) is...
Agrobacterium tumefaciens has two polyphosphate (polyP) kinases, one of which (PPK1AT) is responsible for the formation of polyP granules, while the other (PPK2AT) is used for replenishing the NTP pools by using polyP as a phosphate donor to phosphorylate nucleoside diphosphates. Fusions of eYFP with PPK2AT or of the polyP granule-associated phosin PptA from Ralstonia eutropha always co-localized with polyP granules in A. tumefaciens and allowed the tracking of polyP granules in time-lapse microscopy experiments without the necessity to label the cells with the toxic dye DAPI. Fusions of PPK1AT with mCherry formed fluorescent signals often attached to, but not completely co-localizing with, polyP granules in wild-type cells. Time-lapse microscopy revealed that polyP granules in about one-third of a cell population migrated from the old pole to the new cell pole shortly before or during cell division. Many cells de novo formed a second (nonmigrating) polyP granule at the opposite cell pole before cell division was completed, resulting in two daughter cells each having a polyP granule at the old pole after septum formation. Migration of polyP granules was disordered in mitomycin C-treated or in PopZ-depleted cells, suggesting that polyP granules can associate with DNA or with other molecules that are segregated during the cell cycle.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; Cell Division; Cupriavidus necator; Polyphosphates
PubMed: 35168233
DOI: 10.1159/000521970 -
Journal of Bacteriology Jan 2021The type VI secretion system (T6SS) is a widespread antibacterial weapon capable of secreting multiple effectors for inhibition of competitor cells. Most of the...
The type VI secretion system (T6SS) is a widespread antibacterial weapon capable of secreting multiple effectors for inhibition of competitor cells. Most of the effectors in the system share the same purpose of target intoxication, but the rationale for maintaining various types of effectors in a species is not well studied. In this study, we showed that a peptidoglycan amidase effector in , Tae, cleaves d-Ala--diaminopimelic acid (mDAP) and d-Glu bonds in peptidoglycan and is able to suppress the growth of recipient cells. The growth suppression was effective only under the condition in which cells are actively growing. In contrast, the Tde DNase effectors in the strain possessed a dominant killing effect under carbon starvation. Microscopic analysis showed that Tde triggers cell elongation and DNA degradation, while Tae causes cell enlargement without DNA damage in recipient cells. In a rich medium, harboring only functional Tae was able to maintain competitiveness among and its own sibling cells. Growth suppression and the competitive advantage of were abrogated when recipient cells produced the Tae-specific immunity protein Tai. Given that Tae is highly conserved among strains, the combination of Tae and Tde effectors could allow to better compete with various competitors by increasing its survival during changing environmental conditions. The T6SS encodes multiple effectors with diverse functions, but little is known about the biological significance of harboring such a repertoire of effectors. We reported that the T6SS antibacterial activity of the plant pathogen can be enhanced under carbon starvation or when recipient cell wall peptidoglycan is disturbed. This led to a newly discovered role for the T6SS peptidoglycan amidase Tae effector in providing a growth advantage dependent on the growth status of the target cell. This is in contrast to the Tde DNase effectors that are dominant during carbon starvation. Our study suggests that combining Tae and other effectors could allow to increase its competitiveness among changing environmental conditions.
Topics: Agrobacterium tumefaciens; Anti-Bacterial Agents; Bacterial Proteins; Cell Wall; Deoxyribonucleases; Escherichia coli; Peptidoglycan; Type VI Secretion Systems
PubMed: 33168638
DOI: 10.1128/JB.00490-20 -
Advances in Genetics 2022Several species of the genus represent unique bacterial pathogens able to genetically transform plants, by transferring and integrating a segment of their own DNA... (Review)
Review
Several species of the genus represent unique bacterial pathogens able to genetically transform plants, by transferring and integrating a segment of their own DNA (T-DNA, transferred DNA) in their host genome. Whereas in nature this process results in uncontrolled growth of the infected plant cells (tumors), this capability of has been widely used as a crucial tool to generate transgenic plants, for research and biotechnology. The virulence of relies on a series of virulence genes, mostly encoded on a large plasmid (Ti-plasmid, tumor inducing plasmid), involved in the different steps of the DNA transfer to the host cell genome: activation of bacterial virulence, synthesis and export of the T-DNA and its associated proteins, intracellular trafficking of the T-DNA and effector proteins in the host cell, and integration of the T-DNA in the host genomic DNA. Multiple interactions between these bacterial encoded proteins and host factors occur during the infection process, which determine the outcome of the infection. Here, we review our current knowledge of the mechanisms by which bacterial and plant factors control virulence and host plant susceptibility.
Topics: Virulence; Agrobacterium tumefaciens; Plants, Genetically Modified; Plasmids; Bacteria; Bacterial Proteins; Virulence Factors
PubMed: 37283660
DOI: 10.1016/bs.adgen.2022.08.001 -
Current Topics in Microbiology and... 2018The Agrobacterium tumefaciens VirB/VirD4 translocation machine is a member of a superfamily of translocators designated as type IV secretion systems (T4SSs) that... (Review)
Review
The Agrobacterium tumefaciens VirB/VirD4 translocation machine is a member of a superfamily of translocators designated as type IV secretion systems (T4SSs) that function in many species of gram-negative and gram-positive bacteria. T4SSs evolved from ancestral conjugation systems for specialized purposes relating to bacterial colonization or infection. A. tumefaciens employs the VirB/VirD4 T4SS to deliver oncogenic DNA (T-DNA) and effector proteins to plant cells, causing the tumorous disease called crown gall. This T4SS elaborates both a cell-envelope-spanning channel and an extracellular pilus for establishing target cell contacts. Recent mechanistic and structural studies of the VirB/VirD4 T4SS and related conjugation systems in Escherichia coli have defined T4SS architectures, bases for substrate recruitment, the translocation route for DNA substrates, and steps in the pilus biogenesis pathway. In this review, we provide a brief history of A. tumefaciens VirB/VirD4 T4SS from its discovery in the 1980s to its current status as a paradigm for the T4SS superfamily. We discuss key advancements in defining VirB/VirD4 T4SS function and structure, and we highlight the power of in vivo mutational analyses and chimeric systems for identifying mechanistic themes and specialized adaptations of this fascinating nanomachine.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; Mutagenesis; Recombinant Fusion Proteins; Type IV Secretion Systems
PubMed: 29808338
DOI: 10.1007/82_2018_94 -
Microbiology Spectrum Mar 2019The bacterial type IV secretion systems (T4SSs) are a functionally diverse superfamily of secretion systems found in many species of bacteria. Collectively, the T4SSs... (Review)
Review
The bacterial type IV secretion systems (T4SSs) are a functionally diverse superfamily of secretion systems found in many species of bacteria. Collectively, the T4SSs translocate DNA and monomeric and multimeric protein substrates to bacterial and eukaryotic cell types. T4SSs are composed of two large subfamilies, the conjugation machines and the effector translocators that transmit their cargoes through establishment of direct donor-target cell contacts, and a third small subfamily capable of importing or exporting substrates from or to the milieu. This review summarizes recent mechanistic and structural findings that are shedding new light on how T4SSs have evolved such functional diversity. Translocation signals are now known to be located C terminally or embedded internally in structural folds; these signals in combination with substrate-associated adaptor proteins mediate the docking of specific substrate repertoires to cognate VirD4-like receptors. For the Dot/Icm system, recent work has elucidated the structural basis for adaptor-dependent substrate loading onto the VirD4-like DotL receptor. Advances in definition of T4SS machine structures now allow for detailed comparisons of nanomachines closely related to the VirB/VirD4 T4SS with those more distantly related, e.g., the Dot/Icm and Cag T4SSs. Finally, it is increasingly evident that T4SSs have evolved a variety of mechanisms dependent on elaboration of conjugative pili, membrane tubes, or surface adhesins to establish productive contacts with target cells. T4SSs thus have evolved extreme functional diversity through a plethora of adaptations impacting substrate selection, machine architecture, and target cell binding.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; Biodiversity; DNA, Bacterial; Helicobacter pylori; Legionella pneumophila; Protein Transport; Substrate Specificity; Type IV Secretion Systems
PubMed: 30953428
DOI: 10.1128/microbiolspec.PSIB-0012-2018 -
Environmental Microbiology Jan 2018Many important pathogens maintain significant populations in highly disparate disease and non-disease environments. The consequences of this environmental heterogeneity... (Review)
Review
Many important pathogens maintain significant populations in highly disparate disease and non-disease environments. The consequences of this environmental heterogeneity in shaping the ecological and evolutionary dynamics of these facultative pathogens are incompletely understood. Agrobacterium tumefaciens, the causative agent for crown gall disease of plants has proven a productive model for many aspects of interactions between pathogens and their hosts and with other microbes. In this review, we highlight how this past work provides valuable context for the use of this system to examine how heterogeneity and transitions between disease and non-disease environments influence the ecology and evolution of facultative pathogens. We focus on several features common among facultative pathogens, such as the physiological remodelling required to colonize hosts from environmental reservoirs and the consequences of competition with host and non-host associated microbiota. In addition, we discuss how the life history of facultative pathogens likely often results in ecological tradeoffs associated with performance in disease and non-disease environments. These pathogens may therefore have different competitive dynamics in disease and non-disease environments and are subject to shifting selective pressures that can result in pathoadaptation or the within-host spread of avirulent phenotypes.
Topics: Agrobacterium tumefaciens; Biofilms; Biological Evolution; Ecology; Plant Tumors; Plants; Plasmids
PubMed: 29105274
DOI: 10.1111/1462-2920.13976