Journal of Plant Pathology (2014), 96 (1), 57-62 Anil et al. 57 LEUCINE ZIPPER-LIKE MOTIFS OF HRPZ PSS ARE NOT ESSENTIAL TO INDUCE HYPERSENSITIVE RESPONSE IN TOBACCO K. Anil, B.V. Raju and A.R. Podile Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad-500 046, India SUMMARY Harpins are heat-stable, glycine-rich proteins secreted by several Gram-negative phytopathogenic bacteria, in- volved in the induction of hypersensitive response in non- host plants. HrpZ Pss is highly heat stable, exists as a poly- dispersed, multimeric protein in nature and the functional significance for its existence in many oligomeric forms, remains still unclear. It was suggested that the leucine zipper-like motifs (LZMs) may take part in the formation of oligomeric aggregates, which may be responsible for HR elicitation by harpins and for their high thermal stability. To test the involvement of LZMs in HR elicitation and induction of defense responses, we have generated deletion mutants with or without LZMs and analysed their ability to induce HR, and/or activate the defense responses. The deletion mutant HrpZMM1, with no LZM also elicited HR as well as defense responses in tobacco suggesting the LZMs are not essential for elicitor activity of HrpZ Pss . However, the ability to elicit defense responses may be linked with the ability to induce HR. Key words: HrpZ Pss , Hypersensitive response, truncated mutations. INTRODUCTION Harpins are proteinaceous elicitors of defense and hypersensitive response (HR) in a wide range of crop plants, produced by plant pathogenic bacteria like Pseu- domonas, Erwinia, Xanthomonas and Ralstonia. HrpZ Pss from Pseudomonas syringae pv. syringae retains its bio- logical activity in several deletion mutants, at different sites, (Alfano et al., 1996) in an unique manner. The ac- tivation of different responses by harpin was assigned to different structural features. Intact HrpZ pph is required for the formation of ion-conducting pores, whereas for the induction of defense responses, only its C-terminal part is crucial (Engelhardt et al., 2009). Sequence analy- sis of HrpZ Pss revealed the presence of at least two pos- sible leucine-zipper-like motifs (LZMs) which were also found in other harpins (Tarafdar et al., 2009). LZMs are located on helices present on protein surfaces, signifying that they can take part in the formation of oligomeric ag- gregates, which may be responsible for HR elicitation by harpins and for their high thermal stability (Tarafdar et al ., 2009). Random and site directed mutagenesis of HpaG, from Xanthomonas axonopodis pv . glycines enabled the identification of a region essential for elicitor activity, consisting of a peptide 23 amino acid (aa) in size (H 2 N- NQGISEKQLDQLLTQLIMALLQQ-COOH) (Kim et al., 2004). This region shared a homology of 78 and 74% with 23 and 27 aa regions of the HrpW of Pseudomonas and Erwinia spp, respectively. The stretch of 12 highly hydrophilic aa (H 2 N-QGISEKQLDQLL-COOH) that partially overlaps the N-terminal α-helical regions of the respective proteins is critical for the elicitation of HR in tobacco by Hpa1 Xoo and Hpa1 Xoc . Further, the coiled coil integrity of the harpin is important for dimerization and HR elicitation in tobacco as revealed by two single mis- sense mutants Hpa1 Xoo (L51P) and Hpa1 Xoc (L53P) (Wang et al., 2008). Although these aa are crucial for elicitation of HR in tobacco, they are not necessarily involved in the induction of systemic acquired resistance against Tobacco mosaic virus (TMV) (Wang et al., 2008), which reveals that the dual roles of harpin require different structural determinants. HrpZ Pss has a single tryptophan (W167) residue, which makes it a protein of special interest for fluorescence quenching studies. HrpZ Pss sequence analysis revealed its dissimilarity in aa sequence (He et al ., 1993; Engelhardt et al ., 2009) and lack of homology with any known protein in databases, which makes it a unique biological molecule for functional or structural studies. Although HrpZ Pss was biochemically characterized, and also analyzed by muta- tional analysis, its high thermal stability was not addressed from a structural point of view. Circular dichroism (CD) analysis revealed that the protein is predominantly α-helical (51.5%) and contains a high percentage (13.5%) of leucine (He et al ., 1993). The primary structure of HrpZ Pss con- tains heptadic repeats of leucine and other hydrophobic aa (leucine zipper-like motif; LZM). Two possible LZMs were found in the region of 73-87 (219-261 base pairs, bp) and 245-266 (735-798 bp) aa stretch of HrpZ Pss . Compa- rable leucine-zipper-like motifs were also predicted in eight other harpins (Tarafdar et al ., 2009) and one more possible heptadic LZM was identified in the 299-334 (897-1002 bp) aa stretch. LZMs in most of the harpins are composed by Edizioni ETS Pisa, 2014 Corresponding author: A.R. Podile Fax: +91.40.23010120. E-mail: arpsl@uohyd.ernet.in