Transcriptome analysis reveals key roles of AtLBR-2 in LPS-induced defense responses in plants

Abstract

Background: Lipopolysaccharide (LPS) from Gram-negative bacteria cause innate immune responses in animals and plants. The molecules involved in LPS signaling in animals are well studied, whereas those in plants are not yet as well documented. Recently, we identified Arabidopsis AtLBR-2, which binds to LPS from Pseudomonas aeruginosa (pLPS) directly and regulates pLPS-induced defense responses, such as pathogenesis-related 1 (PR1) expression and reactive oxygen species (ROS) production. In this study, we investigated the pLPS-induced transcriptomic changes in wild-type (WT) and the atlbr-2 mutant Arabidopsis plants using RNA-Seq technology.

Results: RNA-Seq data analysis revealed that pLPS treatment significantly altered the expression of 2139 genes, with 605 up-regulated and 1534 down-regulated genes in WT. Gene ontology (GO) analysis on these genes showed that GO terms, "response to bacterium", "response to salicylic acid (SA) stimulus", and "response to abscisic acid (ABA) stimulus" were enriched amongst only in up-regulated genes, as compared to the genes that were down-regulated. Comparative analysis of differentially expressed genes between WT and the atlbr-2 mutant revealed that 65 genes were up-regulated in WT but not in the atlbr-2 after pLPS treatment. Furthermore, GO analysis on these 65 genes demonstrated their importance for the enrichment of several defense-related GO terms, including "response to bacterium", "response to SA stimulus", and "response to ABA stimulus". We also found reduced levels of pLPS-induced conjugated SA glucoside (SAG) accumulation in atlbr-2 mutants, and no differences were observed in the gene expression levels in SA-treated WT and the atlbr-2 mutants.

Conclusion: These 65 AtLBR-2-dependent up-regulated genes appear to be important for the enrichment of some defense-related GO terms. Moreover, AtLBR-2 might be a key molecule that is indispensable for the up-regulation of defense-related genes and for SA signaling pathway, which is involved in defense against pathogens containing LPS.

Keywords: Arabidopsis; Defense response; Lipopolysaccharide; Plant immunity; RNA-Seq; Salicylic acid.

Fig. 1

The number of differentially expressed genes in pLPS-treated WT plants. Each RNA-Seq data set obtained from untreated WT, pLPS-treated atlbr-2-1, and untreated atlbr-2-1 plants, were compared with that obtained from pLPS-treated WT plants. The numbers in the parentheses indicate the number of genes identified as up-regulated (a) or down-regulated (b) in the pLPS-treated WT plants. The numbers of genes, which were up- or down-regulated only in pLPS-treated WT but not in the other three conditions, are indicated in bold type

Fig. 2

GO classification of pLPS-responsive up-regulated genes. BP GO terms obtained from the GO analysis of 605 pLPS-induced up-regulated genes in WT are shown with the blue line. The same analysis conducted with 540 genes, which excluded 65 AtLBR-2-dependent up-regulated genes from the 605 genes, are shown with the red line. The red font highlights no enriched GO terms in the 540 genes. An arrow indicates the GO term identified only in 540 genes. Scale of y axis shows the percentage of genes that are annotated for each biological process. P < 0.01

Fig. 3

qRT-PCR analysis of six AtLBR-2-dependent up-regulated genes. Among the 65 pLPS-induced AtLBR-2-dependent up-regulated genes, 6 defense-related genes (CYP71A13, LURP1, PNP-A, AIG1, GSTF7, and PDR12) were randomly selected and analyzed by qRT-PCR in WT and atlbr-2-1 plants treated with pLPS. The mean expression values were calculated from the results of three independent experiments. Means ± standard errors are presented. Significant differences among the means were determined by two-way ANOVA followed by post hoc Bonferroni test compared to WT plants; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Comparison of RNA-Seq results with those of qRT-PCR. The Log₂FC values for transcript levels observed in RNA-Seq data (white bar) of randomly selected 20 genes, including AtLBR-2-dependent -independent up- or down-regulated genes, were compared to the results obtained from qRT-PCR (gray bar). AtLBR-2-dependent up- or down-regulated genes are indicated with asterisk: CYP71A13, LURP1, PNP-A, AIG1, GSTF7, PDR12, UNE11, and AT3G20340. AtLBR-2-independent up- or down-regulated genes are indicated without asterisk: HVA22B (AT5G62490), HVA22 homologue B; PDF1.3 (AT2G26010), plant defensin 1.3; CLE21 (AT5G64800), clavata3/ESR-related 21; HAI2 (AT1G07430), highly ABA-induced 2; LEA4–5 (AT5G06760), late embryogenesis abundant 4–5; HR2 (AT3G50460), homolog of RPW8 2; ATH2 (AT3G47740), A. thaliana ABC2 homolog 2; MES13 (AT1G26360), methyl esterase 13; M17 (AT2G41260), late embryogenesis abundant gene; PR12 (AT1G75830); PP2-A6 (AT5G45080), phloem protein 2-A6; PROPEP3 (AT5G64905), elicitor peptide 3 precursor

Fig. 5

pLPS-induced free SA and SAG accumulation and SA-induced gene expression. (a) Quantification of the total levels of free SA and SAG were measured by HPLC using samples extracted from Arabidopsis treated with pLPS for the indicated time. (b) Among the 65 pLPS-induced AtLBR-2-dependent up-regulated genes, the expression of the 3 SA-related genes (PR1, LURP1, and PDR12) were analyzed by qRT-PCR in WT and the atlbr-2-1 mutants plants treated with SA. The mean values were calculated from the results of three independent experiments. Means ± standard errors are presented. Significant differences among the means were determined by two-way ANOVA followed by post hoc Bonferroni test compared to WT plants; *P < 0.05, **P < 0.01. FW, fresh weight