ABSTRACT
Chloroplast precursor proteins encoded in the nucleus depend on their targeting sequences for delivery to chloroplasts. There exist different routes to the chloroplast outer envelope, but a common theme is the involvement of molecular chaperones. Hsp90 (heat-shock protein 90) delivers precursors via its receptor Toc64, which transfers precursors to the core translocase in the outer envelope. In the present paper, we identify an uncharacterized protein in Arabidopsis thaliana OEP61 which shares common features with Toc64, and potentially provides an alternative route to the chloroplasts. Sequence analysis indicates that OEP61 possesses a clamp-type TPR (tetratricopeptide repeat) domain capable of binding molecular chaperones, and a C-terminal TMD (transmembrane domain). Phylogenetic comparisons show sequence similarities between the TPR domain of OEP61 and those of the Toc64 family. Expression of mRNA and protein was detected in all plant tissues, and localization at the chloroplast outer envelope was demonstrated by a combination of microscopy and in vitro import assays. Binding assays show that OEP61 interacts specifically with Hsp70 (heat-shock protein 70) via its TPR clamp domain. Furthermore, OEP61 selectively recognizes chloroplast precursors via their targeting sequences, and a soluble form of OEP61 inhibits chloroplast targeting. We therefore propose that OEP61 is a novel chaperone receptor at the chloroplast outer envelope, mediating Hsp70-dependent protein targeting to chloroplasts.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Chloroplasts/metabolism , HSP70 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Plastids/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Blotting, Western , DNA, Plant/genetics , HSP70 Heat-Shock Proteins/genetics , Molecular Chaperones/genetics , Molecular Sequence Data , Phylogeny , Protein Binding , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Plant/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
Previous analysis of transcriptional changes after elicitation of Cf-9 transgenic tobacco (Nicotiana tabacum) by Avr9 peptide revealed a rapidly upregulated gene, ACRE276. We show that ACRE276 is transiently induced in wounded leaves within 15 min, but upon Avr9 elicitor treatment, this upregulation is enhanced and maintained until cell death onset in Cf-9 tobacco. ACRE276 RNA interference (RNAi) silencing in tobacco results in loss of hypersensitive response (HR) specified by Cf resistance genes. ACRE276 RNAi plants are also compromised for HR mediated by the tobacco mosaic virus defense elicitor p50. Silencing tomato (Lycopersicon esculentum) ACRE276 leads to breakdown of Cf-9-specified resistance against Cladosporium fulvum leaf mold. We confirmed that tobacco ACRE276 is an E3 ubiquitin ligase requiring an intact U-box domain. Bioinformatic analyses revealed Arabidopsis thaliana PLANT U-BOX17 (PUB17) and Brassica napus ARC1 as the closest homologs of tobacco ACRE276. Transiently expressing PUB17 in Cf-9 tobacco silenced for ACRE276 restores HR, while mutant PUB17 lacking E3 ligase activity fails to do so, demonstrating that PUB17 ligase activity is crucial for defense signaling. Arabidopsis PUB17 knockout plants are compromised in RPM1- and RPS4-mediated resistance against Pseudomonas syringae pv tomato containing avirulence genes AvrB and AvrRPS4, respectively. We identify a conserved class of U-box ARMADILLO repeat E3 ligases that are positive regulators of cell death and defense across the Solanaceae and Brassicaceae.
