Genetic Manipulation of <i>Agrobacterium</i>

Morton, Elise R.; Fuqua, Clay

doi:10.1002/9780471729259.mc03d02s25

Cited by 50 publications

(93 citation statements)

References 11 publications

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“…All expression vectors were transformed into C58C1, C58C1ΔpopZ, and C58C1 ΔpopZ::mchy-popZ (12) strains, as indicated, using standard electroporation conditions (36).…”

Section: Methodsmentioning

confidence: 99%

Absence of the Polar Organizing Protein PopZ Results in Reduced and Asymmetric Cell Division in Agrobacterium tumefaciens

et al. 2017

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Agrobacterium tumefaciens is a rod-shaped bacterium that grows by polar insertion of new peptidoglycan during cell elongation. As the cell cycle progresses, peptidoglycan synthesis at the pole ceases prior to insertion of new peptidoglycan at midcell to enable cell division. The A. tumefaciens homolog of the Caulobacter crescentus polar organelle development protein PopZ has been identified as a growth pole marker and a candidate polar growth-promoting factor. Here, we characterize the function of PopZ in cell growth and division of A. tumefaciens. Consistent with previous observations, we observe that PopZ localizes specifically to the growth pole in wild-type cells. Despite the striking localization pattern of PopZ, we find the absence of the protein does not impair polar elongation or cause major changes in the peptidoglycan composition. Instead, we observe an atypical cell length distribution, including minicells, elongated cells, and cells with ectopic poles. Most minicells lack DNA, suggesting a defect in chromosome segregation. Furthermore, the canonical cell division proteins FtsZ and FtsA are misplaced, leading to asymmetric sites of cell constriction. Together, these data suggest that PopZ plays an important role in the regulation of chromosome segregation and cell division.IMPORTANCE A. tumefaciens is a bacterial plant pathogen and a natural genetic engineer. However, very little is known about the spatial and temporal regulation of cell wall biogenesis that leads to polar growth in this bacterium. Understanding the molecular basis of A. tumefaciens growth may allow for the development of innovations to prevent disease or to promote growth during biotechnology applications. Finally, since many closely related plant and animal pathogens exhibit polar growth, discoveries in A. tumefaciens may be broadly applicable for devising antimicrobial strategies.KEYWORDS Agrobacterium, PopZ, cell polarity, chromosome segregation, cell division, growth polarity A grobacterium tumefaciens, the causative agent of crown gall disease in flowering plants, has been studied extensively with regard to pathogenesis and its ability to transfer engineered DNA to plant cells using the type IV secretion system encoded by multiple vir genes. More recent observations of A. tumefaciens growth have revealed that this bacterium exhibits polar growth during elongation (1, 2). In A. tumefaciens, as well as in other Rhizobiales, peptidoglycan is inserted at the new poles created by cell division until the cell doubles in length. The peptidoglycan synthesis is then directed to midcell to enable septum formation and cell division. The growth patterning of A.

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“…All expression vectors were transformed into C58C1, C58C1ΔpopZ, and C58C1 ΔpopZ::mchy-popZ (12) strains, as indicated, using standard electroporation conditions (36).…”

Section: Methodsmentioning

confidence: 99%

Absence of the Polar Organizing Protein PopZ Results in Reduced and Asymmetric Cell Division in Agrobacterium tumefaciens

et al. 2017

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“…All the custom-made DNA was made by Integrated DNA Technologies, Inc. The artificial attachment site was introduced into the tetRA locus using allelic replacement as described previously (16). A synthetic double-stranded 373-bp gBlock gene fragment, att miniTn7 tet locus, was ligated into pMEH0125 with the NdeI and NcoI restriction endonuclease sites.…”

Section: Methodsmentioning

confidence: 99%

“…Nonpolar, markerless deletions of the A. tumefaciens rem (Atu0573), uppX (Atu0480), and ctrA (Atu2434) genes were generated using the plasmids pNPTS138⌬rem, pNPTS138⌬uppX, and pNPTS138⌬ctrA and following an established allelic-replacement protocol (16). Primary integrants were made by conjugation with E. coli S17-1 cells as the donor strain for pNPTS138/139.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, we engineered an a-attTn7 site at a cryptic tetracycline locus in the A. tumefaciens genome (Fig. 1B) using a standard allelic-exchange protocol (16). Replacement of the tetRA locus with a-attTn7 and subsequent integration of a Tn7 cassette did not impact growth, motility, or biofilm formation of A. tumefaciens ( Fig.…”

Section: Construction Of a Tumefaciens With An A-atttn7 Site Enablesmentioning

confidence: 99%

“…tumefaciens is a genetically tractable bacterium, and current methods exist for the construction of transposon mutants and deletion of nonessential genes (16). A plasmid-based complementation system using a reengineered lac promoter is available and has been shown to provide tight regulation of target genes (17).…”

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Mini-Tn 7 Insertion in an Artificial att Tn 7 Site Enables Depletion of the Essential Master Regulator CtrA in the Phytopathogen Agrobacterium tumefaciens

Figueroa-Cuilan

Daniel

Howell

et al. 2016

Appl Environ Microbiol

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Mechanistic studies of many processes in Agrobacterium tumefaciens have been hampered by a lack of genetic tools for characterization of essential genes. In this study, we used a Tn7-based method for inducible control of transcription from an engineered site on the chromosome. We demonstrate that this method enables tighter control of inducible promoters than plasmidbased systems and can be used for depletion studies. The method enables the construction of depletion strains to characterize the roles of essential genes in A. tumefaciens. Here, we used the strategy to deplete the alphaproteobacterial master regulator CtrA and found that depletion of this essential gene results in dramatic rounding of cells, which become nonviable. IMPORTANCEAgrobacterium tumefaciens is a bacterial plant pathogen and natural genetic engineer. Thus, studies of essential processes, including cell cycle progression, DNA replication and segregation, cell growth, and division, may provide insights for limiting disease or improving biotechnology applications. Members of the genus Agrobacterium are common soil-dwelling bacteria. Most are saprophytic and survive primarily by consuming decaying organic material; but some cause phytopathic diseases, including Agrobacterium rhizogenes (hairy root disease), Agrobacterium rubi (cane gall disease), Agrobacterium vitis (crown gall of grape) and Agrobacterium tumefaciens (crown gall disease). During infection, A. tumefaciens transfers a portion of its DNA (transfer DNA [T-DNA]) to plants, resulting in the formation of crown galls in susceptible flowering plants. Infected plants form tumors that limit transport of water and nutrients, leading to decreased vigor and crop yield (1, 2). Because of the ubiquity of A. tumefaciens and its ability to evade plant defenses, crown gall disease continues to be a problem, with documented economic impact on fruit and nut trees, particularly in nurseries (2). Thus, A. tumefaciens has become a model for the study of host-pathogen interactions (3-5) and processes related to pathogenicity, including type IV secretion (6), quorum sensing (7), and biofilm formation (8). Finally, mechanistic studies of A. tumefaciens-mediated DNA transfer into host cells have been harnessed for the genetic engineering of plants, fungi, and mammals (5, 9-11).While the processes related to phytopathogenicity have been subject to intense scrutiny, processes essential for A. tumefaciens survival in the environment, both in the rhizosphere and in association with hosts, are poorly understood. Recent studies have shown that A. tumefaciens cells exhibit a striking pattern of polar cell growth (12-14), leading to a vast array of experimental questions (15). Proper spatial and temporal regulation of the cell cycle must be required in order to coordinate key processes, such as chromosome replication and segregation, cell wall biogenesis, and cell division. Understanding the mechanisms underlying these essential processes will provide key insights into the basic biology of A. tumefaciens...

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Luciferase Complementation Assay for Protein‐Protein Interactions in Plants

Zhou

2018

CP Plant Biology

123

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Constitutive and dynamic protein-protein interactions are fundamental to all aspects of cellular processes. Compared to other techniques measuring protein-protein interactions in plants, the luciferase complementation assay has a number of advantages: it detects plant protein-protein interactions in real time, requires little hands-on manipulation of samples, is highly quantitative, has extremely low background, and can be easily scaled up for high-throughput interactome studies. Here, we describe a protocol that includes two alternate data collection methods to quantify luminescence results based on Agrobacterium-mediated transient luciferase expression in Nicotiana benthamiana. One data collection method employs a charge-coupled device imaging system that allows the interactions to be presented as images, and the other employs a luminometer, which enables the assay to be conducted in a 96-well plate. Technical parameters related to frequently encountered problems and common errors, presented here, are important for performing this assay successfully. © 2018 by John Wiley & Sons, Inc.

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Genetic Manipulation of Agrobacterium

Cited by 50 publications

References 11 publications

Absence of the Polar Organizing Protein PopZ Results in Reduced and Asymmetric Cell Division in Agrobacterium tumefaciens

Absence of the Polar Organizing Protein PopZ Results in Reduced and Asymmetric Cell Division in Agrobacterium tumefaciens

Mini-Tn 7 Insertion in an Artificial att Tn 7 Site Enables Depletion of the Essential Master Regulator CtrA in the Phytopathogen Agrobacterium tumefaciens

Luciferase Complementation Assay for Protein‐Protein Interactions in Plants

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