Identification of Peroxisome-Targeted Proteins Implicated in Plant Innate Immunity in Arabidopsis thaliana

Abstract

Peroxisomes are subcellular organelles, traditionally known to be involved in processes like photorespiration, fatty acid β-oxidation, and detoxification of reactive oxygen species. Proteome analysis of plant peroxisomes and targeting signal prediction methods are important tools to identify novel peroxisomal proteins. In the present study the accuracy of newly developed methods to predict peroxisome targeting signals type 1 (PTS1) in plant proteins was investigated by in vivo subcellular targeting analyses. Upon application of these prediction methods to the Arabidopsis thaliana genome, 392 gene models were predicted to possess functional PTS1 domains, several proteins of which were validated as peroxisomal and numerous novel PTS1 tripeptides were identified. Furthermore, several detoxification-related enzymes and defense-related Arabidopsis proteins were detected by proteome analyses and PTS1 prediction methods that were potentially targeted to peroxisomes.

Two enzymes of the ascorbate-glutathione (ASC-GSH) cycle, glutathione reductase 1 (GR1) and dehydroascorbate reductase 1 (DHAR1), and five glutathione-S transferases (GSTs) had been detected by proteome analysis in leaf peroxisomes. In vivo subcellular localization targeting analyses of the present study verified peroxisomal targeting for GR1 and the protein was found to carry a functional novel PTS1 (TNL>). By contrast, the four GSTs remained cytosolic in the chosen orientation in the back of the reporter protein. New fragmented evidence has been emerging in the literature for an important role of plant peroxisomes in innate immunity. In the present study sixteen defense-related Arabidopsis proteins were experimentally investigated for protein targeting to peroxisomes by in vivo subcellular localization. The proteins of interest included several yet unknown homologs of Arabidopsis NDR1 and tobacco HIN1, the so-called NDR1/HIN1 like (NHL) proteins. In vivo subcellular localization was primarily investigated for three NHL family members (NHL4, NHL6 and NHL25). Peroxisome targeting was verified for NHL4 with strong indications also for NHL6 and NHL25 in being located in peroxisomes. AtIAN12 is a homolog of AIG1/AtIAN8 and had been identified by Arabidopsis leaf peroxisome proteomics. In vivo subcellular localization experiments demonstrated that AtIAN12 protein is targeted to peroxisomes and indicated that the targeting pathway involves posttranslational protein modification by isoprenylation. Taken together, the data indicate for the first time that one NDR1/HIN1 homolog (NHL4) and AtIAN homolog (AtIAN12) are peroxisome associated. Preliminary gene expression analyses indicated that three NHL genes and three AtIAN genes are induced by a bacterial pathogen (Pst DC3000), while NHL6, NHL25, and AtIAN8 are induced by an avirulent Pst DC3000 strain (carrying the effector avrRpt2). Out of the six NHL and AtIAN genes, only NHL6 appeared to be induced in wt Col-0 plants by the bacterial elicitor (flg22), but remained unaffected in Arabidopsis plants carrying a mutation in the flagellin receptor gene FLS2. The data suggested that NHL6 is involved in basal PAMP triggered immunity (PTI). Furthermore, NHL6 transcripts accumulated similarily in both wt plants and npr1 mutant plants after flg22 treatment, which indicates that NHL6 induction is NPR1-independent. Functional studies were initiated through the isolation of homozygous mutants, amiRNA lines and overexpresser lines for selected NHL and AtIAN genes. In homozygous mutants (three nhl mutants and ian11), differences in bacterial proliferation were observed compared to wt plants upon infection with the avirulent bacterium Pst DC3000 (avrRpt2). Overall, the identification of several defense-related proteins in peroxisomes together with preliminary functional data on NHL proteins opens new perspectives to important, multi-layered peroxisome functions in plant innate immunity.