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PNAS 103 (19): 7460-7464

Copyright © 2006 by the National Academy of Sciences.

GABA controls the level of quorum-sensing signal in Agrobacterium tumefaciens

Romain Chevrot*, Ran Rosen{dagger},{ddagger}, Elise Haudecoeur*, Amélie Cirou*, Barry J. Shelp§, Eliora Ron{dagger}, and Denis Faure*,

*Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, Avenue de la Terrasse, Gif-sur-Yvette 91 198, France; {dagger}Department of Molecular Microbiology and Biotechnology and {ddagger}The Maiman Institute for Proteome Research, Tel Aviv University, Tel Aviv 69978, Israel; and §Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada N1G 2W1


Figure 1
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Fig. 1. Expression of the A. tumefaciens operon attKLM in the presence of GABA. (a) The images show part of the two-dimensional gels around the expected position of AttK (indicated by an arrow), which was detected only in A. tumefaciens wild type in the presence of GABA (2 mM). (b) beta-Galactosidase activity (in Miller units) was measured in cultures of A. tumefaciens C58 (attK::lacZ) 2 h after addition of the compounds tested (1 mM) Suc, succinate; {alpha}-KG, {alpha}-ketoglutarate. Values are given as means ± SD; black bars indicate means that are significantly different from that obtained with the addition of water as a blank control (Student’s t test with P < 0.05). (c) Structures of GABA, SSA, GHB, GBL, and OC8-HSL.

 

Figure 2
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Fig. 2. Requirement of GABA transporter Bra for the attKLM expression in A. tumefaciens. (a) Growth of the A. tumefaciens wild type (squares) and the mutant braE (triangles) in the presence of GABA as the sole nitrogen source. (b and c) The expression of the attK::lacZ fusion (beta-galactosidase activity) in cultures of A. tumefaciens wild type (filled symbols in b and filled bars in c) and braE mutant (open symbols in b or open bars in c) was measured (in b) 2 h after the addition of SSA (triangles) or GABA (squares) and (in c) 20 h after the addition of GABA or wounded 6-week-old tomato stems (WS) (0.25 g of fresh weight per milliliter). (d) GABA and Glu concentrations in unwounded (S) and wounded (WS) 6-week-old tomato stems; values are given as means ± SD.

 

Figure 3
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Fig. 3. Effect of GABA on the concentration of OC8-HSL in A. tumefaciens cultures at the end of the exponential growth phase. (a) The OC8-HSL concentration was measured in cultures of A. tumefaciens C58 wild type (squares) and its {Delta}(attJKLM) derivative (triangles) overexpressing (open symbols) or not overexpressing (filled symbols) the traR gene. The dashed line indicates the detection limit of OC8-HSL in this experiment. (b) Under the same conditions described in a, the expression of the attK::lacZ (open squares) and attJ::lacZ (filed squares) fusions and the cell density [in colony-forming units (CFU) per milliliter; filled circles] of the A. tumefaciens C58 cultures were measured. Values are given as means ± SD.

 

Figure 4
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Fig. 4. Virulence assays on a wild-type tobacco plant and its transgenic derivatives GAD3, GAD7, GAD{Delta}C1, and GAD{Delta}C2. (a) Virulence assay on disks of tobacco leaves (n = 16) inoculated with A. tumefaciens C58. The scale of symptoms (0- 4) is described in Methods. (b) Virulence assays on stem of whole plants inoculated with A. tumefaciens C58 wild type or its attM mutant. The fresh weight (in grams) of tumors from infected stems of whole plants (n = 8) is given. (a and b) Values are given as means ± SD. Values on the same horizontal line that do not possess the same letter in superscript (a, b, or c) are statistically different (Student’s t test with P < 0.05). (c and d) Photographs illustrating the virulence assays described in b with wild-type, GAD{Delta}C1, and GAD{Delta}C2 tobacco plants infected with A. tumefaciens C58 (c) or attM mutant (d).

 

Figure 5
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Fig. 5. Scheme of attKLM regulation in the presence of wounded plant tissues. (Upper) The stress-induced synthesis and degradation of GABA in plants (6, 7). (Lower) Summary of the knowledge on the catabolic and QS signal-silencing functions of attKLM operon (14, 15), as well as its induction in the presence of SSA, GHB, and GBL (15) and GABA. {alpha}-KG, {alpha}-ketoglutarate; TCA cycle, tricarboxylic acid cycle; SSADH, SSA dehydrogenase; GABA-T, GABA transaminase; Suc, succinate; OC8-HS, N-(3-oxooctanoyl)homoserine.

 

Figure 6
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Fig. 6. Model illustrating control of vir and QS pathways by plant signals. In the wild-type (a) and GAD{Delta}C transgenic plant lines (b), pathways activated by acetosyringone (AS) and opines are in bold face and that repressed by GABA is in italics; the QS pathway, which is simultaneously activated and repressed in the particular case of the GAD{Delta}C transgenic plant lines, is in bold italics.

 


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