Figure 1.
Pie chart representing percent ratios of putative function-based groups of the tobacco genes activated during PTI.
156 putatively PTI-activated tobacco genes were found by suppression subtractive hybridization of samples taken 6 and 48 hours after inoculation of tobacco leaves with P. syringe hrcC- mutant bacteria. BLAST nucleotide similarity search (Altschul et al. 1990) was used to assign putative functions to the sequences. Based on the identified putative functions the genes were classified into 15 groups: cell structure (2), signaling (20), cellular protection (7), cell wall (11), phenylpropanoid (9), terpenoid (4), defense related (16), heat shock (9), protein metabolism (13), transport (12), metabolism and energy (21), photosynthesis (2), miscellaneous (7), unknown function (11), no significant similarity (32). The ratios and the corresponding percentages are demonstrated in the figure.
Table 1.
cDNA microarray hybridization results of contigs identified from PTI-induced tobacco.
Figure 2.
Validation of microarray results by real time RT-PCR.
A) Heat map diagram of the expression ratios of randomly selected genes obtained by microarray hybridization and real time RT-PCR. Tobacco leaves were infiltrated with P. syringae pv. syringae hrcC- bacteria suspension or water, samples were taken 6 and 48 hours later. Values are averages of three independent biological replicates. Stars indicate significant gene activation greater than 2-fold (p<0.05) as compared to water treated controls. Color coding: red: activation, green: repression, yellow: no significant activity change. The letter “n” means no data available. B) Correlation of the RT-PCR and microarray derived expression ratios of the genes from part A). RT-PCR ratios (both 6 and 48 hpi) from the table in Fig. 2A) were drawn on the y axis, while array derived ratios were drawn on the x axis of the graph. Both axes are on a logarithmic scale. The best fitting regression trend line was found to be an order 2 polynomial regression trend line having the highest r-squared (R2) value with p<0.01.
Figure 3.
Verification of gene-activation by real-time RT-PCR.
A) signal transduction-related genes. B) phenylpropanoid pathway and lignification genes. Ratios of mRNA levels compared to water-treated controls in the order of the strength of activation at 6 hours after treatment. Tobacco leaves were infiltrated with P. syringae pv. syringae hrcC-suspension, samples were taken 6 and 48 hours later. Values are averages of three independent biological replicates. Each replicate was normalized by corresponding actin levels. Stars indicate significant gene activation (p<0.05) as compared to water treated controls. Contig C171 (ubiquitin extension protein) was used as a constitutive control. C) Flagellin as an elicitor of the phenylpropanoid pathway and lignification genes. Ratios of mRNA levels compared to water-treated controls are indicated. Tobacco leaves were infiltrated with flagellin 22 peptide solution, samples were taken 3 and 6 hours later. Values are averages of three independent biological replicates. Each replicate was normalized by corresponding actin levels. Stars indicate significant gene activation (p<0.05) as compared to water treated controls. Legend A) C139: sphingosine-1-phosphate lyase (SPL); C66: SR1 Nt-rab7b membrane-associated GTP-binding protein; C59: CND41, chloroplast nucleoid DNA binding protein; C94: phosphatidylinositol synthase; C106: putative PTS HPR protein involved in serine-phosphorilation; C24: NtMKP1 MAP kinase; C90: receptor-like protein kinase; C108: ethylene forming enzyme (EFE); C125: annexin-like protein; C144: WD repeat protein AN11; C55: 14-3-3 protein isoform; C135: DNA binding protein Rav; C53: calcium-dependent protein kinase 4 CDPK4; C58: MAP3K-like protein kinase; C151: NtHSF1 heat shock transcription factor; C95: Avr9/Cf-9 rapidly elicited protein 31 (ACRE31). Legends B) and C) PAL, Phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxycinnamoyl-CoA ligase; OMT I, O-methyltransferase; POX, peroxidase; F5H, ferulate 5-hydroxylase.
Figure 4.
A current view of the phenylpropanoid metabolism.
Colours indicate the enzymes found in our PTI cDNA library. Each colour corresponds to a specific type of enzyme. Numbers of contigs, and KEGG codes of each enzyme are indicated (Kyoto Encyclopedia of Genes and Genomes http://www.genome.ad.jp/kegg/). Dashed arrows indicate that the process mechanism of the given enzymatic reaction is not known. Abbreviations: PAL, Phenilalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; MO, monophenol oxidase; COMT I, caffeic/5-hydroxyferulic acid O-methyltransferase; F5H, ferulate 5-hydroxylase; 4CL, 4-hydroxycinnamoyl-CoA ligase; C3H, p-coumarate 3-hydroxylase; HCT, hydroxycinnamoyl-transferase; CCoAOMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl-alcohol dehydrogenase; POX, peroxidase; CHS, chalcone-synthase, CHI, chalchone-isomerase; B2H, benzoic acid-2-hydroxylase. (Based on [54])
Figure 5.
HR-inhibition tests and electrolyte leakage measurements to show the ability of PIP to reduce PTI efficiency.
A) Representative images showing the difference in the extension of the HR lesion induced by P. syringae 61 challenge inoculation following pretreatment with P. syringae hrcC- bacteria (hrcC), verus pretreatment with PIP+hrcC, and controls. B) Numerical evaluation of the data. Whole intervein areas were pretreated with piperonylic acid (PIP), P. syringae hrcC- bacteria (hrcC), their combination (PIP+hrcC), or as controls: with dimethyl sulfoxide (DMSO), water (W) or left untreated (C). Challenge treatments with P. syringae 61 (HR-inducing wild type) bacteria followed after 4, 5 and 6 hours. The area of the challenge infiltration was circumscribed with a marker pen. The percentage of this area where HR developed, was noted. Values are the average of three independent experiments. Standard deviations are included in the diagram, and stars indicate significant difference (p<0.05) as compared to the hrcC-treatment. C) Electrolyte leakage measurements to quantify degree of HR development. P. syringae hrcC- bacteria (hrcC) alone or in combination with piperonylic acid (PIP+hrcC) were injected into tobacco leaves. 5 and 6 hours after the pre-treatment, leaves were injected with P. syringae pv. syringae 61 (HR-inducing wild type). Leaf disks were cut out 16 hours later, floated on double distilled H2O, and conductivity was measured in a time series. Significant differences between hrcC and PIP+hrcC conductivity values are indicated by stars (p<0.05).
Figure 6.
Changes in cinnamic acid and p-coumaric acid metabolite levels, and transcript levels of the related PPP enzymes in PTI-induced, PIP inhibitor-treated, and control tobacco leaf tissue.
A) Thin Layer Chromatography (TLC) separation and densitometric measurement of two phenolic compounds, cinnamic acid and p-coumaric acid in PTI-induced and control tobacco leaf tissue. Tobacco leaves were treated with dimethyl sulfoxide (DMSO), piperonylic acid (PIP), P. syringae hrcC- bacteria (hrcC), their combination (PIP+hrcC), flagellin 22 peptide (flg22) and combination of piperonylic acid and flagellin 22 peptide (PIP+flg22). Tissue samples (500 mg) were collected 6 hours after infiltration. Separation and detection of p-coumaric acid and cinnamic acid was carried out by thin layer chromatography (TLC) and UV densitometry. A standard line was created using commercially available p-coumaric acid and cinnamic acid to quantitate the two substances in the measured samples. Standard deviations are included in the diagram, and stars indicate significant difference between PIP+PTI elicitors (PTI+hrcC or PTI+flg22) versus elicitor only treatments (hrcC or flg22 respectively) (p<0.05). B) Measurement of gene-activation of phenylpropanoid pathway and lignification genes by real-time RT-PCR. Tobacco leaves were treated with dimethyl sulfoxide (DMSO), piperonylic acid (PIP), P. syringae hrcC- bacteria (hrcC), their combination (PIP+hrcC), flagellin 22 peptide (flg22) and combination of piperonylic acid and flagellin 22 peptide (PIP+flg22). Tissue samples (500 mg) were collected 3 hours after infiltration. Ratios of mRNA levels compared to water-treated controls are indicated. Values are averages of three replicates. Each replicate was normalized by corresponding actin levels. Stars indicate significant difference between DMSO control and PIP; furthermore between elicitor only treatments (hrcC or flg22) versus PIP+PTI elicitors (PTI+hrcC or PTI+flg22 respectively) (p<0.05). PAL, Phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxycinnamoyl-CoA ligase; OMT I, O-methyltransferase; POX, peroxidase; F5H, ferulate 5-hydroxylase.