Transformation and regeneration of English elm using wild-type Agrobacterium tumefaciens

Nicotiana alata Link and Otto. was transformed via Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase (IPT) that catalyzes cytokinin synthesis. This was considered an autoregulatory senescence-inhibitor system. In 1996, we reported delayed senescence of intact flowers by 2 to 6 d and delayed leaf senescence of transgenic vs. wild-type N. alata. Further evaluations in 1997 revealed several other interesting effects of the SAG12-IPT gene construct. Measurement of chlorophyll content of mature leaves showed higher levels of both chlorophyll a and b in transgenic material under normal fertilization and truncated fertilization regimes. At 4 to 5 months of age transgenic plants expressed differences in plant height, branching, and dry weight. Plant height was reduced by 3 to 13 cm; branch counts increased 2 to 3 fold; and shoot dry weight increased up to 11 g over wild-type N. alata. These observations indicate the system is not tightly autoregulated and may prove useful to the floriculture industry for producing compact and more floriferous plants.

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Extracellular Glycanases of Rhizobium leguminosarum Are Activated on the Cell Surface by an Exopolysaccharide-Related Component

Journal of Bacteriology ◽

10.1128/jb.182.5.1304-1312.2000 ◽

2000 ◽

Vol 182 (5) ◽

pp. 1304-1312 ◽

Cited By ~ 27

Author(s):

Angeles Zorreguieta ◽

Christine Finnie ◽

J. Allan Downie

Keyword(s):

Cell Wall ◽

Agrobacterium Tumefaciens ◽

Cell Surface ◽

Rhizobium Leguminosarum ◽

Plant Cell Wall ◽

Agar Medium ◽

Wild Type ◽

Close Proximity ◽

Mutant Strains ◽

Colony Surface

ABSTRACT Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies of R. leguminosarum, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMC degradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS+ strain, indicating that EPS is required for activation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer of PlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on a lawn ofR. leguminosarum. This indicates that the surface ofA. tumefaciens is inappropriate to activate CMC degradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.

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Root Colonization by Agrobacterium tumefaciens Is Reduced in cel, attB,attD, and attR Mutants

Applied and Environmental Microbiology ◽

10.1128/aem.64.7.2341-2345.1998 ◽

1998 ◽

Vol 64 (7) ◽

pp. 2341-2345 ◽

Cited By ~ 52

Author(s):

Ann G. Matthysse ◽

Susan McMahan

Keyword(s):

Agrobacterium Tumefaciens ◽

Root Length ◽

Root Colonization ◽

Bacterial Pathogenesis ◽

Bacterial Suspension ◽

Cellulose Synthesis ◽

Wild Type ◽

Tomato Roots ◽

Extensive Growth ◽

Number Of Bacteria

ABSTRACT Root colonization by Agrobacterium tumefaciens was measured by using tomato and Arabidopsis thaliana roots dipped in a bacterial suspension and planted in soil. Wild-type bacteria showed extensive growth on tomato roots; the number of bacteria increased from 103 bacteria/cm of root length at the time of inoculation to more than 107 bacteria/cm after 10 days. The numbers of cellulose-minus and nonattachingattB, attD, and attR mutant bacteria were less than 1/10,000th the number of wild-type bacteria recovered from tomato roots. On roots of A. thalianaecotype Landsberg erecta, the numbers of wild-type bacteria increased from about 30 to 8,000 bacteria/cm of root length after 8 days. The numbers of cellulose-minus and nonattaching mutant bacteria were 1/100th to 1/10th the number of wild-type bacteria recovered after 8 days. The attachment of A. tumefaciens to cut A. thaliana roots incubated in 0.4% sucrose and observed with a light microscope was also reduced with cel andatt mutants. These results suggest that cellulose synthesis and attachment genes play a role in the ability of the bacteria to colonize roots, as well as in bacterial pathogenesis.

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Spermidine Inversely Influences Surface Interactions and Planktonic Growth in Agrobacterium tumefaciens

Journal of Bacteriology ◽

10.1128/jb.00265-16 ◽

2016 ◽

Vol 198 (19) ◽

pp. 2682-2691 ◽

Cited By ~ 15

Author(s):

Yi Wang ◽

Sok Ho Kim ◽

Ramya Natarajan ◽

Jason E. Heindl ◽

Eric L. Bruger ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Biofilm Formation ◽

Direct Synthesis ◽

Wild Type ◽

Cyclic Diguanylate ◽

Content Type ◽

Exogenous Addition ◽

In The Wild ◽

Growth Requirement ◽

Transposon Insertions

ABSTRACTIn bacteria, the functions of polyamines, small linear polycations, are poorly defined, but these metabolites can influence biofilm formation in several systems. Transposon insertions in an ornithine decarboxylase (odc) gene inAgrobacterium tumefaciens, predicted to direct synthesis of the polyamine putrescine from ornithine, resulted in elevated cellulose. Null mutants forodcgrew somewhat slowly in a polyamine-free medium but exhibited increased biofilm formation that was dependent on cellulose production. Spermidine is an essential metabolite inA. tumefaciensand is synthesized from putrescine inA. tumefaciensvia the stepwise actions of carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase (CASDC). Exogenous addition of either putrescine or spermidine to theodcmutant returned biofilm formation to wild-type levels. Low levels of exogenous spermidine restored growth to CASDH and CASDC mutants, facilitating weak biofilm formation, but this was dampened with increasing concentrations. Norspermidine rescued growth for theodc, CASDH, and CASDC mutants but did not significantly affect their biofilm phenotypes, whereas in the wild type, it stimulated biofilm formation and depressed spermidine levels. Theodcmutant produced elevated levels of cyclic diguanylate monophosphate (c-di-GMP), exogenous polyamines modulated these levels, and expression of a c-di-GMP phosphodiesterase reversed the enhanced biofilm formation. Prior work revealed accumulation of the precursors putrescine and carboxyspermidine in the CASDH and CASDC mutants, respectively, but unexpectedly, both mutants accumulated homospermidine; here, we show that this requires a homospermidine synthase (hss) homologue.IMPORTANCEPolyamines are small, positively charged metabolites that are nearly ubiquitous in cellular life. They are often essential in eukaryotes and more variably in bacteria. Polyamines have been reported to influence the surface-attached biofilm formation of several bacteria. InAgrobacterium tumefaciens, mutants with diminished levels of the polyamine spermidine are stimulated for biofilm formation, and exogenous provision of spermidine decreases biofilm formation. Spermidine is also essential forA. tumefaciensgrowth, but the related polyamine norspermidine exogenously rescues growth and does not diminish biofilm formation, revealing that the growth requirement and biofilm control are separable. Polyamine control of biofilm formation appears to function via effects on the cellular second messenger cyclic diguanylate monophosphate, regulating the transition from a free-living to a surface-attached lifestyle.

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Agrobacterium tumefaciens Transformation of the Radiation Hypersensitive Arabidopsis thaliana Mutants uvh1 and rad5

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.11.1136 ◽

1998 ◽

Vol 11 (11) ◽

pp. 1136-1141 ◽

Cited By ~ 19

Author(s):

Jaesung Nam ◽

Kirankumar S. Mysore ◽

Stanton B. Gelvin

Keyword(s):

Arabidopsis Thaliana ◽

Agrobacterium Tumefaciens ◽

Transformation Process ◽

Stable Transformation ◽

Wild Type ◽

Dna Integration ◽

Agrobacterium Tumefaciens Transformation ◽

Inoculation Assay

The Arabidopsis thaliana mutants uvh1 and rad5, originally identified as radiation hypersensitive, were reported to be deficient in T-DNA integration based on the relative efficiencies of stable transformation and T-DNA transfer. We reassessed these mutants for susceptibility to transformation by Agrobacterium tumefaciens. The mutant rad5 showed a significant reduction in the efficiency of transient as well as stable transformation, compared with its wild-type progenitor. These data indicate that rad5 is blocked at a step in the transformation process prior to T-DNA integration. We additionally found, using both an in vitro root inoculation and an in vivo flower bolt inoculation assay, that the mutant uvh1 is as susceptible to A. tumefaciens-mediated transformation as is its wild-type progenitor, C10.

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Attachment to Roots and Virulence of a chvB Mutant of Agrobacterium tumefaciens Are Temperature Sensitive

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.2.160 ◽

2002 ◽

Vol 15 (2) ◽

pp. 160-163 ◽

Cited By ~ 13

Author(s):

Ryan Bash ◽

Ann G. Matthysse

Keyword(s):

Arabidopsis Thaliana ◽

Agrobacterium Tumefaciens ◽

Lycopersicon Esculentum ◽

Daucus Carota ◽

Room Temperature ◽

Tagetes Erecta ◽

Temperature Sensitive ◽

Wild Type

Agrobacterium tumefaciens chvB mutants are unable to produce β-1,2 glucan. They are nonattaching and avirulent and show reduced motility at room temperature. At lower temperatures (16°C), chvB mutants became virulent on Bryophyllum daigremontiana and Lycopersicon esculentum and were able to attach to L. esculentum, Arabidopsis thaliana, Daucus carota, and Tagetes erecta roots. The mutant bacteria also recovered wild-type motility at lower temperatures. Two other nonattaching mutants of A. tumefaciens, AttR and AtrA, were unaffected by the lowered temperature, remaining nonattaching and avirulent.

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Agrobacterium tumefaciens VirB6 Protein Participates in Formation of VirB7 and VirB9 Complexes Required for Type IV Secretion

Journal of Bacteriology ◽

10.1128/jb.185.9.2867-2878.2003 ◽

2003 ◽

Vol 185 (9) ◽

pp. 2867-2878 ◽

Cited By ~ 74

Author(s):

Simon J. Jakubowski ◽

Vidhya Krishnamoorthy ◽

Peter J. Christie

Keyword(s):

Agrobacterium Tumefaciens ◽

Protein Complexes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Type Iv Secretion ◽

Wild Type ◽

Mutant Proteins ◽

Type Iv ◽

Inner Membrane Protein ◽

Substrate Transfer

ABSTRACT This study characterized the contribution of Agrobacterium tumefaciens VirB6, a polytopic inner membrane protein, to the formation of outer membrane VirB7 lipoprotein and VirB9 protein multimers required for type IV secretion. VirB7 assembles as a disulfide cross-linked homodimer that associates with the T pilus and a VirB7-VirB9 heterodimer that stabilizes other VirB proteins during biogenesis of the secretion machine. Two presumptive VirB protein complexes, composed of VirB6, VirB7, and VirB9 and of VirB7, VirB9, and VirB10, were isolated by immunoprecipitation or glutathione S-transferase pulldown assays from detergent-solubilized membrane extracts of wild-type A348 and a strain producing only VirB6 through VirB10 among the VirB proteins. To examine the biological importance of VirB6 complex formation for type IV secretion, we monitored the effects of nonstoichiometric VirB6 production and the synthesis of VirB6 derivatives with 4-residue insertions (VirB6.i4) on VirB7 and VirB9 multimerization, T-pilus assembly, and substrate transfer. A virB6 gene deletion mutant accumulated VirB7 dimers at diminished steady-state levels, whereas complementation with a plasmid bearing wild-type virB6 partially restored accumulation of the dimers. VirB6 overproduction was correlated with formation of higher-order VirB9 complexes or aggregates and also blocked substrate transfer without a detectable disruption of T-pilus production; these phenotypes were displayed by cells grown at 28°C, a temperature that favors VirB protein turnover, but not by cells grown at 20°C. Strains producing several VirB6.i4 mutant proteins assembled novel VirB7 and VirB9 complexes detectable by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two strains producing the D60.i4 and L191.i4 mutant proteins translocated IncQ plasmid and VirE2 effector protein substrates in the absence of a detectable T pilus. Our findings support a model that VirB6 mediates formation of VirB7 and VirB9 complexes required for biogenesis of the T pilus and the secretion channel.

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Characteristics of morphogenetic responses of Nicotiana langsdorffii on transformation with wild type strains of Agrobacterium in vitro

Ecological genetics ◽

10.17816/ecogen5321-24 ◽

2007 ◽

Vol 5 (3) ◽

pp. 21-24 ◽

Cited By ~ 1

Author(s):

Tatyana V Matveeva ◽

Tatiana Yu Pigichka ◽

Ludmila A Lutova

Keyword(s):

Agrobacterium Tumefaciens ◽

Shoot Formation ◽

Wild Type ◽

Model Object ◽

Nicotiana Langsdorffii ◽

Type Strains ◽

Morphogenetic Responses ◽

Leaf Disks

Ability to transformation by wt strains of Agrobacterium tumefaciens (T37, C58, A6) and A. rhizogenes (15834, 8196, A4) was characterized for Nicotiana langsdorffii. It was shown that effectivity of transformation of this species by strains T37, A6,15834, 8196, A4 was lower comparing to the model object N. tabacum. Tumors induced by A. tumefaciens on leaf disks of N. langsdorffii, tend to shoot formation.

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082 Infection and Transformation of Rhododendron by Agrobacterium tumefaciens Strain B6

HortScience ◽

10.21273/hortsci.34.3.455c ◽

1999 ◽

Vol 34 (3) ◽

pp. 455C-455 ◽

Cited By ~ 1

Author(s):

R.R. Tripepi ◽

M.W. George ◽

T. Sripo ◽

S.A. Johnsen ◽

A.B. Caplan

Keyword(s):

Agrobacterium Tumefaciens ◽

Genetically Modified ◽

Green Fluorescence Protein ◽

Plant Genome ◽

Wild Type ◽

Bacterial Strains ◽

Green Fluorescence ◽

Physical Presence ◽

Fluorescence Protein ◽

Polymerase Chain

The objective of this study was to determine if selected strains of Agrobacterium could infect microshoots of Rhododendron catawbiense. Fifteen microshoot stems of R. catawbiense var. album `America', `Joe Paterno', and `Cunningham's White' were inoculated with two drops (about 25 μL) of wild type Agrobacterium tumefaciens strains C58 or B6 or with wild type A. rhizogenes strain E8/73. Five control shoots were inoculated with 1.2 mM KH2PO4 buffer. Microshoots were grown on woody plant medium (WPM) supplemented with 4.9 μM 2iP. Six weeks after inoculation galls that formed were excised from the microshoots and placed on WPM that lacked plant growth regulators but contained 300 mg·L-1 cefotaxime. In another study, these wild-type bacterial strains were genetically modified by inserting the pBINm-gfp5-ER plasmid, which contained genes coding for NPTII and green fluorescence protein (GFP), into the bacteria. These modified strains were inoculated on 15 stems of the three rhododendron cultivars and one variety. Calluses that formed were excised, placed on basal WPM with cefotaxime, and allowed to proliferate. Wild type C58 induced galls to form on `Joe Paterno', R.c. album, and `Cunningham's White' stems, whereas wild type B6 caused galls to form only on the latter two types of rhododendron. Wild-type E8/73 failed to induce gall formation on the rhododendrons. Only genetically modified B6 caused galls to form on only `Cunningham's White' microshoots (seven of 15 inoculated stems). Three of these galls fluoresced green under ultraviolet light. Physical presence of the NPTII and GFP genes in the plant genome was determined by polymerase chain reaction. This study demonstrated that R. catawbiense is susceptible to Agrobacterium infection, and this plant can be genetically transformed.

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Functional Analysis of the Agrobacterium tumefaciens T-DNA Transport Pore Protein VirB8

Journal of Bacteriology ◽

10.1128/jb.183.12.3636-3641.2001 ◽

2001 ◽

Vol 183 (12) ◽

pp. 3636-3641 ◽

Cited By ~ 25

Author(s):

Renu B. Kumar ◽

Anath Das

Keyword(s):

Agrobacterium Tumefaciens ◽

Point Mutations ◽

Essential Amino Acids ◽

Integral Membrane Protein ◽

Tumor Formation ◽

Dna Transfer ◽

Amino Acid Residues ◽

Wild Type ◽

Protein Protein Interaction ◽

Pcr Method

ABSTRACT The VirB8 protein of Agrobacterium tumefaciens is essential for DNA transfer to plants. VirB8, a 237-residue polypeptide, is an integral membrane protein with a short N-terminal cytoplasmic domain. It interacts with two transport pore proteins, VirB9 and VirB10, in addition to itself. To study the role of these interactions in DNA transfer and to identify essential amino acids of VirB8, we introduced random mutations in virB8 by the mutagenic PCR method. The putative mutants were tested for VirB8 function by the ability to complement a virB8 deletion mutant in tumor formation assays. After multiple rounds of screening 13 mutants that failed to complement the virB8 deletion mutation were identified. Analysis of the mutant strains by DNA sequence analysis, Western blot assays, and reconstruction of new point mutations led to the identification of five amino acid residues that are essential for VirB8 function. The substitution of glycine-78 to serine, serine-87 to leucine, alanine-100 to valine, arginine-107 to proline or alanine, and threonine-192 to methionine led to the loss of VirB8 activity. When introduced into the wild-type strain, virB8 S87Lpartially suppressed the tumor forming ability of the wild-type protein. Analysis of protein-protein interaction by the yeast two-hybrid assay indicated that VirB8R107P is defective in interactions with both VirB9 and VirB10. A second mutant VirB8S87L is defective in interaction with VirB9.

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