Arabidopsis LBP/BPI related-1 and -2 bind to LPS directly and regulate PR1 expression.

Plants detect pathogen invasions by recognition of pathogen-associated molecular patterns (PAMPs). PAMP perception induces various defence responses. Lipopolysaccharide (LPS), a primary constituent of the outer membrane of Gram-negative bacteria, is one of the most studied PAMPs. LPS causes defence responses such as generation of nitrogen oxide (NO) and reactive oxygen species (ROS) in Arabidopsis, tobacco, and rice suspension cells1,2,3. LPS also induces expression of pathogenesis-related (PR) genes, which are up-regulated in pathological or stressful situations, in Arabidopsis leaves1. In addition, LPS induces stomatal closure and NO production in guard cells3. Further, investigation of the LPS recognition mechanism is at the forefront in plant innate immunity studies. Recently, Ranf et al. identified that the bulb-type lectin S-domain-1 receptor-like kinase LORE (lipooligosaccharide-specific reduced elicitation) is required for sensing of LPS from Pseudomonas and Xanthomonas species4. However, it is still unclear which molecule(s), including LORE, can directly bind to LPS. Thus, the identification of these molecule(s) will enhance our understanding of the overall LPS recognition system. In mammals, there are two well-studied proteins that directly bind to LPS, LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI). Human LBP (hLBP) and human BPI (hBPI) structurally resemble each other with 45% amino acid sequence identity. Both proteins play important roles in the regulation of defence responses against LPS. Mammalian LPS recognition is orchestrated by several LPS binding proteins, including LBP, BPI, and a membrane protein CD14, which transfers LPS to a mammalian LPS receptor complex TLR4/MD-25,6. LBP, a serum glycoprotein produced principally by hepatocytes, is critical to rapid and effective signal transduction for induction of proinflammatory cytokines, because it facilitates the transfer of LPS to CD14, then to TLR4/MD-2. In addition, BPI, which is a glycoprotein purified from granules of neutrophils, also binds to LPS with higher affinity than LBP5. The binding of BPI to LPS increases the permeability of the bacterial membranes and opsonizes bacteria to enhance phagocytosis by neutrophils5. Another important aspect of BPI is the attenuation of the LPS-induced inflammatory response by competitive inhibition against LBP5. LBP and BPI belong to a protein family called the LBP/BPI/PLUNC (palate, lung, and nasal epithelial clone) superfamily7. PLUNC protein, an abundant secretory product in human nasal lavage fluid, also can bind to LPS and has been shown to suppress the growth of bacteria8,9. Interestingly, LBP/BPI/PLUNC superfamily proteins have been identified in various species including chicken, fish, and oyster10,11,12,13,14,15. This superfamily can be divided into two subfamilies; LBP/BPI and PLUNC. Ovocalyxin-36 (OCX-36) is an abundant eggshell protein of chicken which is related to the PLUNC subfamily. OCX-36 binds to LPS and shows inhibitory activity against growth of Staphylococcus aureus16. LBP/BPI subfamily proteins of oyster Crassostrea gigas (Cg-BPI1 and Cg-BPI2) also display LPS binding and bactericidal activities15,17. Expression of LBP/BPI subfamily genes was induced by bacterial challenge or LPS treatment in various fish such as rainbow trout, Atlantic cod, carp, and ayu11,12,13,14. However, LBP/BPI/PLUNC superfamily proteins have not been characterised in plants. The fact that members of both subfamilies (i.e., hLBP, hBPI, OCX-36 and Cg-BPI) bind to LPS and participate in innate immune responses against potential bacterial invasion motivated the current study to characterize plant proteins belonging to the LBP/BPI/PLUNC superfamily. Here, we characterised two genes of Arabidopsis, AtLBP/BPI related-1 (AtLBR-1) and AtLBP/BPI related-2 (AtLBR-2), which belong to the LBP/BPI subfamily rather than the PLUNC subfamily. Because many LBP/BPI/PLUNC superfamily proteins were characterised by their LPS binding ability, we studied whether AtLBRs can bind to LPS. As the results, the recombinant N-terminal domain of AtLBRs is found to bind to LPS directly. In addition, LPS-treated atlbr mutants showed the defect in immune responses, such as PR1 gene expression and ROS production. Altogether, these results demonstrate the biological importance of LBRs for induction of LPS-triggered defence responses in plants and the functional similarities among LBP/BPI subfamily from various organisms.