Peroxisomal Localization of Benzyl Alcohol O-Benzoyltransferase HSR201 is Mediated by a Non-canonical Peroxisomal Targeting Signal and Required for Salicylic Acid Biosynthesis.

The phytohormone salicylic acid (SA) regulates plant responses to various types of environmental stress, particularly pathogen infections. We previously revealed that the benzyl alcohol O-benzoyltransferase HSR201 was required for pathogen signal-induced SA synthesis, and its overexpression together with NtCNL, encoding a cinnamate-coenzyme A ligase, was sufficient for the production of significant amounts of SA in tobacco. We herein examined the subcellular localization of HSR201 and found that it fused to a yellow fluorescent protein localized in peroxisomes. Most peroxisomal matrix proteins possess peroxisomal targeting signal type-1 (PTS1) located at the extreme C-terminus or PTS2 located at the N-terminus; however, a bioinformatics analysis failed to identify similar signals for HSR201. Deletion and mutation analyses of HSR201 identified one essential (extreme C-terminal Leu460) and three important (Ile455, Ile456 and Ala459) amino acid residues for its peroxisomal localization. The virus-induced gene silencing (VIGS) of PEX5, a PTS1 receptor, but not PEX7, a PTS2 receptor, compromised the peroxisomal targeting of HSR201 in Nicotiana benthamiana. When overexpressed with NtCNL, HSR201 mutants with reduced or non-peroxisomal targeting induced lower SA levels than the wild type; however, these mutations did not affect the protein stability or activity of HSR201. VIGS of the HSR201 homolog compromised pathogen signal-induced SA accumulation in N. benthamiana, which was complemented by the HSR201 wild type, but not the mutant with non-peroxisomal targeting. These results suggest that the peroxisomal localization of HSR201 is mediated by a non-canonical PTS1 and required for SA biosynthesis.