Characterization of a novel bacterial PAMP - elongation factor Tu - and its role in "Arabidopsis thaliana" defense and immunity

Abstract

The discrimination of self and nonself is a primary challenge for all living organisms to detect microbial invasion and to protect and defend against the invader. If a pathogen manages to overcome constitutive barriers, highly specific recognition systems are able to identify common pathogenic signals and activate the innate immune system as a first line of defense. Specialized cells and a circulatory system that is able to spread somatically generated adaptive immune responses to the infection side exist only in animals. Plants lack an adaptive immune system comparable like this, but they independently co-evolved the capability to detect microbial invasions by perception of specific molecules, so called PAMPs (pathogen associated molecular patterns), and subsequent activation of innate immune responses. Flagellin, the major subunit of the bacterial motility organ flagellum (Yonekura, K. et al. 2001), can be regarded as the best characterized bacterial PAMP in plants. Flg22, a synthetic peptide comprising the highly conserved epitope of the flagellin Nterminus, is recognized by the plant cell and is sufficient to activate innate immune responses. In this work based on the model plant Arabidopsis thaliana, experiments with bacterial extracts devoid of elicitor active flagellin, show still the capability to induce a broad set of plant defense reactions as flagellin, suggesting that at least one additional perception system for another elicitor exist. This novel elicitor and the corresponding active site were identified as the first 18-26 amino acids from the Nterminus of bacterial Elongation factor Tu (elf18-elf26). This essential protein, involved in the delivery of aminoacyl-tRNA to the ribosome during the translation process, is highly conserved over all organisms (Appendix 1) and is the most abundant protein in the bacterial cell (Helms, M. K. and Jameson, D. M. 1995). Furthermore it is considered to be the slowest evolving protein (Gaucher, E. A. et al. 2003) containing all characteristics for a classical PAMP (see definition included in General Introduction). Further characterization of the EF-Tu/Arabidopsis thaliana interaction showed that all known plant defense mechanisms are activated upon EFTu elicitation. This includes extra cellular medium alkalinization of suspension cultured cells of Arabidopsis, production of reactive oxygen species (ROS) (Laloi, C. et al. 2004) and increase in the biosynthesis of plant hormone ethylene. Like shown previously for flagellin (Zipfel, C. et al. 2004), pre-treatment of Arabidopsis with elf-peptides led to enhanced resistance against plant pathogenic bacteria Pseudomonas syringae pv. tomato (DC3000). Gene expression changes were analyzed using Affymetrix ATH1 array and about 1000 genes with induced expression after 30-60 minutes treatment with elf-peptides were identified. These genes were congruent with that affected by flg22 treatment (Navarro, L. et al. 2004, Zipfel, C. et al. 2004). The same genes were also found to be induced in the flagellin insensitive receptor mutant fls2 upon elf-treatment. Binding studies with radio labeled elf26-125I-TY show that there is a high affinity binding site for EF-Tu existing in Arabidopsis thaliana, which is saturable, highly specific and independent of FLS2. Crosslinking experiments identified a polypeptide band of 150 kDa as potential binding site for EF-Tu. Strikingly the perception of one PAMP (e.g. flg22) leads to a higher amount of binding sites for the other elicitor (e.g. elf18). Furthermore the perception of EF-Tu activates, like flg22, a MAP kinase-based signaling cascade in nearly identical kinetic. Together this study indicate two independent receptors using a converging signaling cascade that leads to the activation of the plant innate immune system with the same broad array of plant defense reactions.