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.

Electronic supplementary material

   The online version of this article (10.1186/s12864-017-4372-4) contains
   supplementary material, which is available to authorized users.

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

Background

   The endotoxin lipopolysaccharide (LPS), a major component of the outer
   membranes of Gram-negative bacteria, is one of the most studied
   pathogen-associated molecular patterns (PAMPs). The perception of LPS
   triggers various defense responses in plants and animals [[31]1]. In
   plants, LPS-induced defense responses have been well studied; these
   include LPS-induced generation of reactive oxygen species (ROS) and
   nitrogen oxide (NO), salicylic acid (SA) accumulation, expression of
   pathogenesis-related (PR) genes, and stomatal closure [[32]2–[33]5].
   SA, in particular, is an important signal molecule in plant defense.
   The accumulation of SA is involved in local defenses as well as in
   systemic acquired resistance (SAR) [[34]6].

   The LPS recognition mechanism has been well studied in animals. In
   mammals, LPS-binding protein (LBP) and
   bactericidal/permeability-increasing protein (BPI) play important roles
   in the regulation of immune responses against LPS [[35]7]. Although
   both the proteins directly bind to LPS, BPI inhibits whereas LBP
   enhances the binding of LPS to Toll-like receptor 4, a mammalian LPS
   receptor. Recently, we identified two Arabidopsis LBP/BPI-related
   proteins, AtLBR-1 and AtLBR-2 [[36]8]. When we incubated recombinant
   forms of both AtLBR-1 and AtLBR-2 with Pseudomonas aeruginosa LPS
   (pLPS) separately, they exhibited the capability to bind to it
   directly; atlbr mutants showed deficiencies in pLPS-induced PR1 gene
   expression and ROS generation. We predicted that AtLBR-2 would be more
   important than AtLBR-1 in the induction of defense responses to LPS
   because the binding affinity of AtLBR-2 for LPS appeared higher than
   that of AtLBR-1, and AtLBR-2 is located in the apoplastic region.

   In the present study, we investigated the importance of AtLBR-2 in the
   dynamic changes in Arabidopsis transcriptome in response to LPS
   treatment. To achieve this goal, we performed a transcriptome analysis
   using high-throughput mRNA sequencing (RNA-Seq). RNA-Seq analysis using
   WT and the atlbr-2-1 identified 65 AtLBR-2-dependent genes that were
   up-regulated after LPS treatment. These 65 genes appear to be important
   for the enrichment of some defense-related gene ontology (GO) terms.
   Our findings highlight the indispensable role of AtLBR-2 in defense
   signaling mechanism against LPS.

Results

Transcriptomic analysis of P. aeruginosa LPS-responsive genes in WT
Arabidopsis

   To examine and compare the LPS-induced transcriptional changes between
   wild-type (WT) and the atlbr-2-1, we treated them with LPS from P.
   aeruginosa (pLPS); total RNA was extracted and RNA-Seq analysis was
   performed.

   Firstly, we analyzed the pLPS-responsive genes in the WT. The RNA-Seq
   data obtained from untreated WT were compared with that of pLPS-treated
   WT. We observed that the transcript levels of 2139 genes changed
   significantly in pLPS-treated WT. Of these, 605 genes were identified
   as up-regulated genes in pLPS-treated WT (Fig. [37]1a). Moreover, 1534
   genes were identified as down-regulated genes in pLPS-treated WT (Fig.
   [38]1b). We performed gene ontology (GO) analysis of these genes using
   the functional annotation chart of DAVID. The biological process (BP)
   GO classification of the 605 up-regulated genes identified 33 GO terms
   (P < 0.01, Fig. [39]2, blue line) (Additional file [40]1: Table S1),
   including not only defense-related GO terms, but also several metabolic
   processes-related terms. This finding corresponded with the results
   reported from transcriptional analysis on Arabidopsis seedlings treated
   with LPS from Burkholderia cepacia [[41]9]. Defense-related GO terms
   included, “response to bacterium”, “response to SA stimulus”, “response
   to abscisic acid (ABA) stimulus”, “response to jasmonic acid stimulus”,
   “response to ROS”, and “response to wounding”. In contrast, 1534
   down-regulated genes were classified via 43 GO terms (P < 0.01)
   (Additional file [42]1: Table S2). Interestingly, defense-related GO
   terms, other than “response to bacterium”, “response to SA stimulus”,
   “response to ABA stimulus”, were also common in these 43 GO terms.
   These findings suggested that up-regulation, but not down-regulation,
   of genes related to bacterial responses may be a characteristic of
   normal pLPS-induced gene expression. It can also be inferred that SA-
   and ABA-related pathways may be important for the up-regulation, but
   not down-regulation, of genes after pLPS treatment.

Fig. 1.

   Fig. 1
   [43]Open in a new tab

   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.

   Fig. 2
   [44]Open in a new tab

   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

   Furthermore, cellular component (CC) GO analysis showed that 23.0% of
   the 605 up-regulated genes were categorized as “endomembrane system”;
   also, 22.9% and 11.9% of the 1534 down-regulated genes were categorized
   as “endomembrane system” and “intrinsic to membrane”, respectively
   (Additional file [45]1: Table S3). These results indicated that genes
   activated in the membrane-related region were most affected by the pLPS
   treatment.

Identification of AtLBR-2-dependent up- or down-regulated genes

   To elucidate the importance of AtLBR-2 in pLPS-induced transcriptional
   responses, we identified the genes that were up-regulated in an
   AtLBR-2-dependent manner after 24 h of pLPS treatment. We compared each
   of the three RNA-Seq data with that of pLPS-treated WT (Fig. [46]1).
   Furthermore, we studied the genes that were up- or down-regulated only
   in the pLPS-treated WT plants and not in the other 3 data sets; these
   were then identified as AtLBR-2-dependent up- or down-regulated genes.
   A total of 65 candidate genes were identified to be AtLBR-2-dependent
   up-regulated genes (Fig. [47]1a, Table [48]1). We focused on these 65
   genes and analyzed them further; only two genes, “unfertilized embryo
   sac 11 (UNE11: AT4G00080)” and the gene for an “uncharacterized protein
   (AT3G20340)”, were identified to be AtLBR-2-dependent down-regulated
   genes (Fig. [49]1b).

Table 1.

   AtLBR-2-dependent up-regulated 65 genes after pLPS treatment
   Accession Description Log[2]FC Ref.
   AT2G14610 Pathogenesis-related protein 1 (PR1)* −5.493297 [[50]43]
   AT3G23120 Receptor like protein 38 (RLP38)* −4.553003 [[51]44]
   AT3G21500 1-deoxy-D-xylulose 5-phosphate synthase 1 (DXPS1) −4.454822 ―
   AT2G30770 Putative cytochrome P450 (CYP71A13)* −3.876201 [[52]17]
   AT1G61800 Glucose-6-phosphate/phosphate transporter 2 (GPT2)* −3.440655
   [[53]45, [54]46]
   AT1G21320 Nucleotide binding protein −3.423526 ―
   AT2G14560 Late upregulated in response to Hyaloperonospora parasitica 1
   (LURP1)* −3.420434 [[55]47]
   AT4G35180 LYS/HIS transporter 7 (LHT7)* −3.326263 [[56]44, [57]46]
   AT2G29350 Senescence-associated gene 13 (SAG13)* −3.152003 [[58]48]
   AT4G04510 Cysteine-rich receptor-like kinase (RLK) 38 (CRK38)*
   −3.124063 [[59]49]
   AT2G24850 Tyrosine aminotransferase 3 (TAT3)* −2.935669 [[60]10]
   AT2G18660 Plant natriuretic peptide A (PNP-A)* −2.824188 [[61]50]
   AT5G24200 Alpha/beta-Hydrolases superfamily protein* −2.675765 [[62]51]
   AT2G04070 Multidrug and Toxin Extrusion (MATE) efflux family protein
   −2.651088 ―
   AT3G22235 Pathogen and circadian controlled 1 (PCC1) −2.612637 ―
   AT4G12470 Azelaic acid induced 1 (AZI1)* −2.593354 [[63]52]
   AT1G33960 avrRpt2-induced gene 1 (AIG1)* −2.454032 [[64]53]
   AT1G65500 Uncharacterized protein −2.423526 [[65]45]
   AT4G22470 Lipid transfer protein (LTP) family protein* −2.414268
   [[66]54]
   AT4G12490 Lipid transfer protein* −2.37707 [[67]55]
   AT4G12480 Early Arabidopsis aluminum induced 1 (pEARLI 1)* −2.334243
   [[68]56]
   AT5G46050 Peptide transporter 3 (PTR3)* −2.322650 [[69]57]
   AT3G28580 P-loop containing nucleoside triphosphate hydrolases
   superfamily protein −2.314015 ―
   AT3G50480 Homolog of RPW8 4 (HR4)* −2.311148 [[70]10]
   AT2G26400 Acireductone dioxygenase 3 (ARD3) −2.302582 ―
   AT1G51820 Leucine-rich repeat protein kinase family protein* −2.288417
   [[71]32]
   AT1G02920 Glutathione S-transferase 7 (GSTF7)* −2.267427 [[72]58]
   AT1G65481 Uncharacterized protein −2.255667 ―
   AT1G43910 P-loop containing nucleoside triphosphate hydrolases
   superfamily protein* −2.252226 [[73]44]
   AT4G17660 Protein kinase superfamily protein −2.126580 ―
   AT4G26200 1-Amino-cyclopropane-1-carboxylate synthase (ACS7) −2.091110
   ―
   AT3G63380 Auto-inhibited Ca^2+-ATPase 12 (ACA12)* −2.063108 [[74]24,
   [75]44]
   AT5G09470 Dicarboxylate carriers 3 (DIC3) −2.058293 ―
   AT4G12735 Uncharacterized protein −2.022097 ―
   AT2G25470 Receptor like protein 21 (RLP21) −2.002310 ―
   AT3G26210 Putative cytochrome P450 (CYP71B23) −1.987360 ―
   AT4G23130 Cysteine-rich RLK 5 (CRK5)* −1.985073 [[76]59]
   AT2G25510 Uncharacterized protein* −1.984502 [[77]10]
   AT5G03350 Legume lectin family protein* −1.980512 [[78]10]
   AT3G50770 Calmodulin-like 41 (CLM41)* −1.936221 [[79]55]
   AT4G37990 Elicitor-activated gene 3–2 (ELI3–2)* −1.895395 [[80]60]
   AT4G00170 Plant vesicle-associated membrane protein (VAMP) family
   protein −1.809448 ―
   AT2G19190 Flg22-induced RLK 1 (FRK1)* −1.804904 [[81]61]
   AT2G20720 Pentatricopeptide repeat (PPR) superfamily protein −1.790359
   ―
   AT5G44390 FAD-binding Berberine family protein −1.785875 ―
   AT1G35230 Arabinogalactan-protein 5 (AGP5)* −1.779422 [[82]62]
   AT5G53870 Early nodulin-like protein 1 (ENODL1) −1.774478 ―
   AT2G04050 Multidrug and Toxin Extrusion (MATE) efflux family protein
   −1.766112 ―
   AT1G02930 Glutathione S-transferase 6 (GSTF6)* −1.750494 [[83]63]
   AT2G43620 Chitinase family protein* −1.717382 [[84]64]
   AT1G21250 Cell wall-associated kinase 1 (WAK1)* −1.706512 [[85]10,
   [86]65]
   AT1G80130 Tetratricopeptide repeat (TPR)-like superfamily protein*
   −1.679920 [[87]66]
   AT5G44575 Uncharacterized protein −1.671623 ―
   AT5G62480 Glutathione S-transferase TAU 9 (GSTU9) −1.630394 ―
   AT5G10760 Apoplastic, EDS1-dependent 1 (AED1)* −1.621488 [[88]50]
   AT5G24640 Uncharacterized protein −1.616614 ―
   AT5G64000 3′(2′),5′-bisphosphate nucleotidase (SAL2) −1.600337 ―
   AT3G28540 P-loop containing nucleoside triphosphate hydrolases
   superfamily protein −1.584674 ―
   AT1G05730 Uncharacterized protein (DUF842) −1.542038 ―
   AT1G26420 FAD-binding Berberine family protein −1.526992 [[89]67]
   AT1G67520 Lectin protein kinase family protein −1.478748 ―
   AT2G26440 Pectin methylesterase 12 (PME12)* −1.460767 [[90]68]
   AT3G26830 Phytoalexin deficient 3 (PAD3)* −1.409278 [[91]16, [92]69]
   AT2G41730 Uncharacterized protein −1.405069 ―
   AT1G15520 Pleiotropic drug resistance 12 (PDR12)* −1.361787 [[93]70]
   [94]Open in a new tab

   Genes up-regulated in an AtLBR-2-dependent manner after 24 h pLPS
   treatment were identified (FDR < 0.01, Log[2]FC < −1.35). Genes, which
   were related to plant–pathogen interaction or to SA, are indicated by
   asterisks or in bold type, respectively, with references