The defense‐related rice gene Pir7b encodes an α/β hydrolase fold protein exhibiting esterase activity towards naphthol AS‐esters

Abstract

Acquired resistance of rice to Pyricularia oryzae, the causing agent of rice blast, can be induced by inoculation with the non-host pathogen Pseudomonas syringae pv. syringae. We have previously cloned a cDNA and a corresponding gene (Pir7b) whose transcripts accumulate upon infiltration with the resistance-inducing bacteria. The putative encoded product Pir7b exhibits significant sequence similarity to two recently cloned hydroxynitrile lyases from Manihot esculenta (cassava) and Hevea brasisliensis, enzymes involved in the release of hydrogen cyanide from cyanogenic glycosides. As rice does not contain cyanogenic glycosides, a similar function of Pir7b appears unplausible. In order to functionally characterize the protein, recombinant Pir7b was produced in Escherichia coli and Saccharomyces cerevisiae. We show that recombinant Pir7b does not have hydroxynitrile lyase activity, but exhibits esterase activity towards naphthol AS-acetate. Using Pir7b-specific antibodies, we show that the protein accumulates in rice leaves inoculated with P. syringae pv. syringae. Both the recombinant and the authentic proteins have an apparent molecular mass of 32 kDa (28.8 kDa calculated) and seem to be active as monomers. Pir7b esterase also exhibits sequence similarity to several expressed sequence tags of Arabidopsis thaliana, indicating that it belongs to a family of proteins widely occuring in plants.Keywords : hydroxynitrile lyase; epoxide hydrolase; defense-related esterase; A/β fold.Acquired resistance of rice towards Pyricularia oryzae, the to have diverged from a common ancestor and are characterized by the possession of an A/β hydrolase fold that provides a concausing agent of the rice blast disease, can be triggered by the inoculation of leaves with the non-host pathogen Pseudomonas served arrangement of catalytic residues (the catalytic triad) and a conserved three-dimensional structure even in the absence of syringae pv. syringae [1]. Concomitantly with resistance induction, a set of new transcripts accumulate in the host that are sequence conservation [8]. They catalyze hydrolytic reactions with a wide variety of substrates. Here we report on the heterolothought to play a role in the defense against pathogens. One of the activated genes that was cloned encoded a putative 268-gous expression of the Pir7b cDNA in Escherichia coli and yeast and the isolation and functional characterization of the reamino-acid protein of unknown function that was called Pir7b [2]. A comparison of the Pir7b sequence to entries in recent combinant Pir7b protein. Using a set of potential substrates, we show that recombinant Pir7b is an esterase. Homology and strucdatabase releases revealed significant sequence similarity (35% identical and 56% conserved amino acids; Fig. 1) to two re-tural investigations suggest that Pir7b is a new member of the A/β hydrolase fold structural superfamily. cently cloned A-hydroxynitrile lyases (Hnl) from cassava (Manihot esculenta) [3] and rubber tree (Hevea brasiliensis) [4]. These enzymes catalyze the f...

Metabolic, Genomic, and Biochemical Analyses of Glandular Trichomes from the Wild Tomato SpeciesLycopersicon hirsutumIdentify a Key Enzyme in the Biosynthesis of Methylketones

“…In addition, this peptide is similar to several proteins with no proven function, all considered to be part of the large a/b-hydrolase fold proteins (Waspi et al, 1998;Zhong et al, 2001;Marshall et al, 2003). This cDNA was represented only once (0.04%; accession number AW617364) in the LA1777 gland EST database.…”

“…These include, in addition to PNAE and tomato MJE, potato (Solanum tuberosum) MJE (accession number AY684102) and PIR7, a protein from rice (Oryza sativa) that was shown to have esterase activity with artificial substrates (Waspi et al, 1998). In addition, the tobacco (Nicotiana tabacum) SABP2 protein, which has recently been shown to be methyl salicylate esterase (Forouhar et al, 2005), and an uncharacterized protein encoded by an ethyleneinduced citrus EST (Zhong et al, 2001), as well as 20 proteins encoded in the Arabidopsis genome, show similarity to MKS1 (Figure 12).…”

“…PIR7, PNAE, SABP2, and MJE all appear to have defense-related functions. For example, the defense-related rice Pir7 transcripts were found to accumulate upon infiltration with the resistance-inducing Pseudomonas syringae pv syringae (Waspi et al, 1998), and PNAE is involved in the biosynthesis of defense alkaloids in the Indian medicinal plant R. serpentina. MJE may be involved in the jasmonate signal transduction pathway that turns on many defense genes (Sasaki et al, 2001;Stuhlfelder et al, 2004).…”

Syringolin Reprograms Wheat to Undergo Hypersensitive Cell Death in a Compatible Interaction with Powdery Mildew

“…New products appeared reproducibly in the syringolin A-treated samples, as shown in the examples of autoradiograms in Figures 4A and 4B. In addition, protein gel blot analysis showed that a wheat homolog of the rice Pir7b esterase, which accumulates in rice plants upon syringolin A treatment (Wäspi et al, 1998b), was induced ( Figure 4C). Thus, syringolin A activates host genes in wheat.…”

“…Proteins were separated on 15% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes using standard methods. Blots were incubated using a 1:1000 dilution of an antiserum obtained by immunizing rabbits with a recombinant Pir7b fusion protein (Wäspi et al, 1998b) in combination with a 1:2000 dilution of a secondary alkaline phosphatase-coupled antirabbit IgG antibody (Boehringer Mannheim). Blots were finally developed in a solution containing 3 mM 4-nitroblue tetrazolium chloride,…”