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. 2015 Aug 5;66(21):6697–6714. doi: 10.1093/jxb/erv377

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© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Fig. 4. — Production of DSF is associated with induced callose deposition. (A) Assay for DSF production by different bacterial strains using the Xcc DSF biosensor 8523 (pKLN55) in which the DSF-responsive promoter from the endoglucanase gene is cloned upstream of an eGFP reporter (P_eng:GFP). DSF extracted from the cell-free culture supernatant of Xcc8004 (wild type, DSF⁺); 8523 (DSF^–); Pseudomonas syringae pv. syringae PssB728a (pHM1) (wild-type strain harbouring the empty vector); and PssB728a (pRpfF) (Pss harbouring the Xcc DSF synthetic gene rpfF). An increase in GFP fluorescence (represented on the y-axis) compared with the control (extracts from the Xcc8523; rpfF mutant) indicates the amount of DSF produced by different strains. (B) TLC analysis for the production of DSF and AHL in Xcc and PssB728a. TLC was performed with crude ethyl acetate extracts isolated from the culture supernatant of different strains of Xcc and Pss. DSF (left panel) and AHL (right panel) were detected by an overlay of the Xcc DSF indicator strain 8523 (pKLN55) and the E. coli AHL biosensor (JB524) on TLC plates. The photograph was taken with the UV light source oriented from the top of the TLC plates. Column 1, Xcc8523 (DSF-deficient mutant); column 2, Xcc8004 (Xcc wild-type strain); column 3, PssB728a (pHM1); column 4, PssB728a (pRpfF); column 5, synthetic DSF (20 μM cis-11-methyl-2-dodecenoic acid); column 6, synthetic AHL (10 μM N-3-oxo-hexanoyl-dl-homoserine lactone; 3OC6-HSL). Similar results were obtained in three independent experiments. (C) From left to right, callose deposition in N. bethamiana leaves infiltrated with a 1×10⁶ cfu ml^–1 suspension of different bacterial strains: wild-type Xcc8004, 8523, 8523 (pRpfF), water control, PssB728a (pHM1), and B728a (pRpfF). Callose deposition was visualized by staining with aniline blue and examined using a stereo fluorescence microscope 24h post-inoculation. White dots in these pictures are indicative of callose deposition. (D) Average number of callose deposits per 0.5mm² area. Error bars represent SD values from three leaves of each plant from three independent experiments. Six microscopic fields from each leaf were analysed. Differences between the responses to Xcc8523 (DSF^– mutant; indicated by *P<0.01) and the P. syringae B728a wild-type strain harbouring the Xcc DSF synthase rpfF (indicated by **P<0.001) compared with the wild-type strains of Xcc8004 and P. syringae B728a were significant as assessed by Student’s t-test.