Figure 1.
Transcriptomic analysis in orange leaves inoculated with Xanthomonas citri subsp. citri.
Citrus cDNA microarray hybridization was performed 24 h after inoculation of orange leaves with WT and Δlov strains of X. citri subsp. citri and control treatment (10 mM MgCl2). Three independents biological samples were used. Venn diagrams show the classification of differentially expressed microarray probes according to the following comparisons: X. citri subsp. citri WT-control, X. citri subsp. citri Δlov-control and X. citri subsp. citri WT- X. citri subsp. citri Δlov.
Figure 2.
Evaluation of host photosynthesis during Xanthomonas citri subsp. citri interaction with orange leaves.
(A) Chlorophyll fluorescence parameters were calculated in orange leaves inoculated with the WT and Δlov X. citri subsp. citri strains and control treatments (10 mM MgCl2) at different days post inoculation (dpi): ΦmPSII corresponds to maximum quantum yield of PSII in the dark adapted state (Fv/Fm, Ai), OEmPSII corresponds to maximum operating efficiency of PSII (F’v /F’m, Aii). Fm: maximum chlorophyll fluorescence in dark adapted leaves; Fv: variable fluorescence in dark adapted leaves; Fm’: maximum chlorophyll fluorescence after saturating light pulses, Fv’: variable fluorescence after saturating light pulses. (B) Chlorophyll a (i) and b (ii) contents were measured at different times after orange inoculation with X. citri subsp. citri WT and Δlov strains and control treatment. All results are expressed as the mean of three independent biological replicates and error bars represent the standard deviations. Asterisks indicate significant differences between WT and Δlov treatments (p<0.05).
Figure 3.
CHO metabolism- and defense response-related Citrus ESTs differentially regulated during Xanthomonas citri subsp. citri interaction.
Expression ratios were calculated for genes differentially expressed in orange leaves inoculated with the WT and Δlov X. citri subsp. citri strains and control treatments (10 mM MgCl2, Ctrl) (A) Log2 of expression ratio between treatments (M) for ESTs corresponding to a cell wall invertase and a sucrose synthase (orange1.1g008242m and orange1.1g008531m of Citrus sinensis) (B) Log2 of expression ratio between treatments (M) for ESTs corresponding to two pathogenesis-related proteins [orange1.1g032389m (black columns) and orange1.1g032285m (grey columns) of C. sinensis] and two chitinases [orange1.1g020187m (black columns) and orange1.1g026315m (grey columns) of C. sinensis]. Data are averages of values obtained from three independent biological samples. Bars represent standard error. Accession numbers correspond to the complete C. sinensis genes taken from the phytozome database (http://www.phytozome.net/citrus.php).
Figure 4.
Histological analysis of lignin deposition in orange leaves upon interaction with Xanthomonas citri subsp. citri.
Lignin deposition was analysed by acid fluoroglucin staining of orange leaves inoculated with WT and Δlov X. citri subsp. citri strains and control treatments (10 mM MgCl2). Stained tissues were observed with a visible microscope at 7 days post inoculation (dpi) using a 1000X magnification. The wine-red coloration represents lignin deposition in plant secondary cell walls. 3A panels show infected tissues sections in which xylem vessels as well as phloem and sclerenchymatous fibers surrounding the vascular bundles are visualized. 3B panels show zoomed-in images of sclerenchymatous fibers where internal lignin content is indicated with arrows. Scale bars: 5 µm. Identical results were obtained with three independent biological samples.
Figure 5.
Tissue integrity upon orange interaction with WT and Δlov strains of Xanthomonas citri subsp. citri.
(A) Analysis of log2 of expression ratio between treatments (M) of a Citrus EST corresponding to a phospholipase D (orange1.1g003057m of Citrus sinensis) and an endo-β-mannanase (orange1.1g013811m of C. sinensis). Data are averages of values obtained from three independent biological samples. Bars represent standard error. Accession numbers correspond to the complete C. sinensis genes taken from the phytozome database (http://www.phytozome.net/citrus.php). Ctrl: control treatment (10 mM MgCl2) (B) Ion leakage measurements were performed in bacterial- and control (10 mM MgCl2)-inoculated orange leaves at 2 and 5 days post inoculation (dpi). Results are expressed as percentage of ion leakage and represent the mean of three independent biological replicates. Error bars represent the standard deviations. Asterisks indicate significant differences between WT and Δlov treatments (p<0.05). (C) Structure and organization of inoculated-leaves fragments were analyzed by safranine/fast green staining. Panels show the microscopic visualization of stained tissue fragments 7 days after bacterial and control treatments using a 400X magnification. Scale bars: 10 µm. Identical results were obtained with three independent biological samples, 90% of the optical fields analyzed for each biological replica rendered the same results.