Regulation of<i>Agrobacterium tumefaciens</i>Virulence Gene Expression: Isolation of a Mutation that Restores virGD52E Function

Gubba, Siddeswar; Xie, Yonghong; Das, Anath Bandhu

doi:10.1094/mpmi-8-0788

Cited by 22 publications

(13 citation statements)

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“…These altered proteins contain mutations that converted either asparagine-54 to aspartic acid (virGN54D) or isoleucine-106 to leucine (virGI106L). Both of these mutant proteins stimulated a high level of vir gene expression, especially when expressed from a high-copy-number plasmid (118). When tested in tran-VOL.…”

Section: Virulence Gene Expression and Plant Transformationmentioning

confidence: 99%

Agrobacterium -Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Gelvin

2003

Microbiol Mol Biol Rev

1,169

687

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SUMMARY Agrobacterium tumefaciens and related Agrobacterium species have been known as plant pathogens since the beginning of the 20th century. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this “natural genetic engineer” for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. In this article, I review some of the basic biology concerned with Agrobacterium-mediated genetic transformation. Knowledge of fundamental biological principles embracing both the host and the pathogen have been and will continue to be key to extending the utility of Agrobacterium for genetic engineering purposes.

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Section: Virulence Gene Expression and Plant Transformationmentioning

confidence: 99%

Agrobacterium -Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Gelvin

2003

Microbiol Mol Biol Rev

1,169

687

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“…VirGI77V/D52E was originally discovered as a mutant that restores the activity of VirGD52E (10). In contrast to the low previously reported constitutive activity of virGI77V/D52E (10), driving expression behind the strong P N25 promoter results in superior constitutive activity.…”

Section: Discussionmentioning

confidence: 95%

“…Constitutively active mutants of VirG have been isolated with mutations at three positions, N54 (VirGN54D), I77 (VirGI77V and VirGI77V/D52E), and I106 (VirGI106L, VirGI106F, VirGI106P, and VirGI106Y) (10,11,13,33,36). Among these mutants, only VirGI77V/D52E and VirGN54D are no longer AS responsive, and therefore they are the focus of this investigation.…”

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confidence: 99%

“…It was suggested that the additional negative charge of N54D in the active site may mimic the phosphoryl group in activated VirG (12). However, the conserved D52 residue was still essential for the constitutive activity of VirGN54D, as alleles carrying the D52E mutation resulted in complete loss of activity (10,33). As these alleles appear to have different dependencies on the conserved D52 residue, and possibly distinctive mechanisms, we initiated a more thorough examination of their activities.…”

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confidence: 99%

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Constitutive Activation of Two-Component Response Regulators: Characterization of VirG Activation inAgrobacterium tumefaciens

Gao

Mukhopadhyay²,

Fang

et al. 2006

J Bacteriol

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Response regulators are the ultimate modulators in two-component signal transduction pathways. The N-terminal receiver domains generally accept phosphates from cognate histidine kinases to control output. VirG for example, the response regulator of the VirA/VirG two-component system in Agrobacterium tumefaciens, mediates the expression of virulence genes in response to plant host signals. Response regulators have a highly conserved structure and share a similar conformational activation upon phosphorylation, yet the sequence and structural features that determine or perturb the cooperative activation events are ill defined. Here we use VirG and the unique features of the Agrobacterium system to extend our understanding of the response regulator activation. Two previously isolated constitutive VirG mutants, VirGN54D and VirGI77V/D52E, provide the foundation for our studies. In vivo phosphorylation patterns establish that VirGN54D is able to accumulate phosphates from small-molecule phosphate donors, such as acetyl phosphate, while the VirGI77V/D52E allele carries conformational changes mimicking the active conformation. Further structural alterations on these two alleles begin to reveal the changes necessary for response regulator activation.

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“…The 3Ј flanking sequence were released from pAM1247 as a 2-kb BamHI-BglII fragment; the 5Ј flanking sequence and hphB were released from pAM1248 as a 2.6-kb EcoRIBamHI fragment, and these were cloned into pAM1145 digested with EcoRI and BglII to create pAM1253. The binary vector pAM1145 contains a broad-hostrange origin of replication, the right and left borders of the T-DNA from the octopine Ti-plasmid pTiA6, and a mutated form of virG (virG N54D ), which obviates the need to add compounds such as acetosyringone to induce T-DNA transfer (20). pAM1145 was previously constructed by insertion of a 5.6-kb KpnI-SalI fragment from plasmid pAD1378 (8) into pAD1310 (27).…”

Section: Methodsmentioning

confidence: 99%

Coccidioides posadasii Contains a Single 1,3-β-Glucan Synthase Gene That Appears To Be Essential for Growth

et al. 2005

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1,3-␤-Glucan synthase is responsible for the synthesis of ␤-glucan, an essential cell wall structural component in most fungi. We sought to determine whether Coccidioides posadasii possesses genes homologous to known fungal FKS genes that encode the catalytic subunit of 1,3-␤-glucan synthase. A single gene, designated FKS1, was identified, and examination of its predicted protein product showed a high degree of conservation with Fks proteins from other filamentous fungi. FKS1 is expressed at similar levels in mycelia and early spherulating cultures, and expression decreases as the spherules mature. We used Agrobacterium-mediated transformation to create strains that harbor ⌬FKS1::hygB, a null allele of FKS1, and hypothesize that Fks1p function is essential, due to our inability to purify this allele away from a complementing wild-type FKS1 allele in a heterokaryotic strain. The heterokaryon appears normal with respect to growth rate and arthroconidium production; however, microscopic examination of strains with ⌬FKS1::hygB alleles revealed abnormal swelling of hyphal elements.

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Regulation ofAgrobacterium tumefaciensVirulence Gene Expression: Isolation of a Mutation that Restores virGD52E Function

Cited by 22 publications

References 0 publications

Agrobacterium -Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Agrobacterium -Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Constitutive Activation of Two-Component Response Regulators: Characterization of VirG Activation inAgrobacterium tumefaciens

Coccidioides posadasii Contains a Single 1,3-β-Glucan Synthase Gene That Appears To Be Essential for Growth

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