Plasma Membrane Localization Is Essential for Oryza sativa Pto-Interacting Protein 1a-Mediated Negative Regulation of Immune Signaling in Rice    

Abstract

Oryza sativa Pto-interacting protein 1a (OsPti1a), an ortholog of tomato (Solanum lycopersicum) SlPti1, functions as a negative regulator of innate immunity in rice (Oryza sativa). In ospti1a mutants, the activation of immune responses, including hypersensitive response-like cell death, is caused by loss of the OsPti1a protein; however, it is as yet unclear how OsPti1a suppresses immune responses. Here, we report that OsPti1a localizes to detergent-resistant membrane fractions of the plasma membrane through lipid modification of the protein's amino terminus, which is highly conserved among Pti1 orthologs in several plant species. Importantly, mislocalization of OsPti1a after deletion of its amino terminus reduced its ability to complement the mutant phenotypes, including hypersensitive response-like cell death. Furthermore, complex formation of OsPti1a depends on its amino terminus-mediated membrane localization. Liquid chromatography-tandem mass spectrometry analysis of OsPti1a complexinteracting proteins identified several defense-related proteins. Collectively, these findings indicate that appropriate complex formation by OsPti1a at the plasma membrane is required for the negative regulation of plant immune responses in rice.

Rice immune regulator, OsPti1a, is specifically phosphorylated at the plasma membrane

“…Prior to immunoblotting, the nitrocellulose membrane was analyzed by Coomassie brilliant blue (CBB) staining to confirm protein recovery and equal loading. Gel fractionation methods were described in Matsui et al 7 …”

“…The subcellular fractionation methods were performed as described in Matsui et al 7 To characterize the phosphorylation of OsPti1a protein in the microsomal fraction, rice suspension cultured cells were collected and homogenized in membrane isolation buffer (50 mM Tris-HCl (pH7.5), 0.25M sorbitol, 2 mM EDTA, 5 mM ascorbic acid, 50 mM b-glycerophosphate, 10 mM Na 3 VO 4 , 10 mM NaF, cOmplete Protease Inhibitor Cocktail Tablets [Roche, Mannheim, Germany]). For fractionation analysis, the resulting supernatants were used as the soluble fractions and the pellets were used as the microsomal fractions.…”

“…These findings suggest that the regulation of immune responses mediated by OsPti1a could depend on the interaction and regulation of OsPti1a-associated proteins at the plasma membrane. 7 We reported that upstream kinases, OsOxi1 and OsPdk1, are rapidly phosphorylated upon chitin treatment. 5,6 However, it remains unknown whether the phosphorylation of OsPti1a coincident with its membrane localization.…”

“…OsPti1a was previously shown to specifically localize at the plasma membrane, such that OsPti1a protein in the microsomal fraction came mostly from the plasma membrane. 7 After that, tagged OsPti1a protein (hemagglutinin-strepIItag: HastrepII) was affinity-purified using Strep-Tactin beads. Phosphorylation was detected by Phos-tag molecules that have a high affinity to phosphate groups, thereby shifting the mobility of phosphorylated proteins compared to non-phosphorylated proteins in SDS-PAGE.…”

“…7 Additionally, fractionation analysis and transient expression Keywords: Pti1, plant immunity, phosphorylation, plasma membrane, rice…”