Table 1.
Detailed information on the top up-regulated genes based on RNA-seq analysis (fold change 30 minutes after flg22 and AtPep1 treatments compared to the control).
Genes of interest are highlighted in bold.
Fig 1.
Distribution of differentially expressed genes (DEGs).
(A) DEGs in Arabidopsis thaliana in response to flg22 and AtPep1 treatments compared to the control investigated in this study. Red bars correspond to the up-regulated genes; blue bars correspond to the down-regulated genes. (B) Venn diagram of up-regulated DEGs between flg22 treatment and AtPep1 treatments. (C) Venn diagram of down-regulated DEGs between flg22 treatment and AtPep1 treatments. In B and C, the overlapping regions display the common transcripts. (D) Volcano plot of DEGs in response to flg22 treatment; (E) Volcano plot of DEGs in response to AtPep1 treatment. In (D-E), blue dots correspond to significantly up- and down-regulated DEGs, grey dots represent non-DEGs. At1G56240 (PP2-B13) and At1G69900 (ACLP1) are highlighted in red.
Fig 2.
Confirmation of RNA-seq results by quantitative real-time reverse transcription PCR analysis.
The expression levels of 20 candidate genes in the RNA-Seq transcriptional profile were validated by qRT-PCR using gene-specific primer sets (S3 Table). One-week-old seedlings of Arabidopsis Col-0 plants were treated with 1 μM flg22 and AtPep1 and the expression patterns of the 20 selected candidates were measured 30 minutes after elicitor treatment. The X-axis indicates the genes name; the Y-axis indicates the log2 scale of the gene expression levels. Each bar represents the fold changes relative to mock samples. The relative expression of each gene was normalized to that of ACTIN2 expression. Values were obtained from the means ± SD of three technical replicates. Two independent experiments were performed with the similar results.
Fig 3.
Bacterial susceptibility assay.
(A) Leaves of four- to six-week-old Arabidopsis plants (Col-0, sid2-2, pp2-b13, and aclp1) were pressure infiltrated with Pseudomonas syringae DC3000 hrcC- mutant (OD600 = 0.0002, in infiltration buffer). (B) Leaves of four- to six-week-old Arabidopsis plants (Col-0, sid2-2, pp2-b13, and aclp1) were pressure infiltrated with Pseudomonas syringae pv. tomato DC3000 (OD600 = 0.0002, in infiltration buffer). sid2-2 mutant plants, which are deficient in salicylic acid production, were used as a positive control. Black bars indicate the number of bacterial colony from leaf discs of infected leaves just after infiltration (0 day); white bars represent colony-forming units (cfu/cm2) 48 h post-inoculation. Bars show the mean ± s.e. of six technical replicates. Six plants were used for each line. Similar results were observed in four independent experiments. Asterisks indicate a significant difference (*p-value ≤0.05, **p-value ≤0.01) from the wild-type plants as determined by Student’s t-test.
Fig 4.
Changes in expression levels of the PP2-B13 and ACLP1 genes after elicitor treatment.
Leaf discs of five weeks old Arabidopsis Col-0 plants were treated with 1 μM flg22 and AtPep1 and the expression patterns of the PP2-B13 and ACLP1 genes were measured 30 min, 2 h, and 6 h after elicitor treatment. Expression was measured by quantitative reverse transcription (RT)-PCR using gene-specific primers. The X-axis indicates the genes name; the Y-axis indicates the log2 scale of the gene expression levels. Each bar represents the fold changes relative to mock samples. Data were normalized using the housekeeping gene Ubiquitin. Values were obtained from the means ± SD of three technical replicates and analyzed by Student’s t-test. Two independent experiments were performed with the similar results. P-values are indicated *p-value ≤0.05, **p-value ≤0.01, ***p-value ≤0.001.
Fig 5.
Early PTI responses upon elicitor treatment.
Ethylene accumulation after elicitor treatment. Leaf discs of four- to five-week-old plants of wild-type and mutant lines (pp2-b13, and aclp1) were treated with 1 μM of the flg22 elicitor peptide or without any peptide (control). In all cases, ethylene production was measured three and a half hours after closing the tubes. Ethylene accumulation in pp2-b13 and aclp1 mutant lines was compared to the wild-type Arabidopsis. fls2 mutant line was used as a negative control. Values were obtained from the mean ethylene concentration ± SD of six technical replicates. Similar results were obtained in at least six independent experiments. T-test was performed comparing the responses of the control treatment to the elicitor treatments; P-values are indicated *p-value ≤0.05. Additional repeats are in the S6 Fig (Panels A-C).
Fig 6.
ROS production after treatment with flg22.
Leaf discs were treated with 1 μM flg22 or without any peptide (control). (A) indicates ROS production in pp2-b13 and aclp1 mutant lines compared to wild-type Arabidopsis; (B) represents maximum ROS production in pp2-b13 and aclp1 mutant lines compared to wild-type Arabidopsis. fls2 mutant line was used as a negative control. Graphs display average of 12 technical replicates. Error bars indicate standard error (SE) of the mean. The experiment was repeated four times with similar results. RLU = relative light units. T-test was performed comparing the responses of the control treatment to the elicitor treatments; P-values are indicated *p-value ≤0.05.
Fig 7.
FLS2 protein levels of the mutant lines pp2-b13 and aclp1 as detected by immunoblot using a FLS2-specific antibody. fls2 mutant plant is used as the negative control. Ponceau S staining was used as the loading control. The original gel image is presented in S7 Fig.
Fig 8.
Measurement of free Salicylic acid in pp2-b13, aclp1 and wild-type plants after infiltration with Psuodomonas syringae DC3000 hrcC.
Four- to five-week-old plants of wild-type and mutant lines (pp2-b13, and aclp1) were infiltrated with Pst DC3000 hrcC (OD600 = 0.0002, in infiltration buffer). Fours leaves for each plants were infiltrated. Treated plants with infiltration buffer regarded as Mock-Control. Six plants were used for each replicates and three technical replicates were taken for each experiments. 48-hour post infiltration the leaves were collected and the free SA levels was measured. sid2-2 mutant plants, were used as a control. Bars show the mean ± s.e. of three technical replicates. Two independent experiments were performed with the similar results. Statistical analyses were performed using the Students t-test. P-values are indicated *p-value ≤0.05.
Fig 9.
Expression levels of three major defense marker genes in pp2-b13 and aclp1 mutant lines compared to the wild-type Arabidopsis (Col-0).
Five-week-old plants of wild-type and mutant lines pp2-b13, and aclp1 were pressure infiltrated with Pst DC3000 hrcC (OD600 = 0.0002, in infiltration buffer) and the expression patterns of the genes including PATHOGENESIS-RELATED GENE1 (PR1), VEGETATIVE STORAGE PROTEIN1 (VSP1), and PLANT DEFENSIN1.2 (PDF1.2) were measured 48 hours after infiltration. Expression was measured by quantitative reverse transcription (RT)-PCR using gene-specific primers. Pressure infiltrated plants with infiltration buffer were regarded as Mock-Control. The X-axis indicates the genes name; the Y-axis indicates the gene expression levels. The relative expression of each marker gene was normalized to that of ACTIN2 expression. Each bar represents the fold changes relative to mock samples. Values were obtained from the means ± SD of three technical replicates of pooled leaves harvested from six plants for each line. Three independent experiments were performed with the similar results. P-values are indicated *p-value ≤0.05, **p-value ≤0.01, ***p-value ≤0.001.