Analysis of the role of BONZAI 1/COPINE 1 in Arabidopsis defense responses.

摘要

The copines are a class of newly defined, Ca2+-dependent, phospholipid-binding proteins. Mutations in the Arabidopsis COPINE1 (CPN1, also BONZAI 1/BON1) gene cause a humidity- and temperature-dependent lesion-mimic phenotype and dramatically increased resistance to a bacterial and an oomycetous pathogen. Previous studies suggest that CPN1 functions as a negative regulator of defense responses. The current study was aimed to further analyze CPN1 function through genetic, molecular and cell biological approaches. Fluorescence-tagged CPN1 protein was localized to the plasma membrane (PM) and quickly accumulated at cell junctions and lobes after pathogen attack. Structural-functional studies showed that transgenic expression of the CPN1 VWA domain caused a lesion-mimic phenotype that partially phenocopied cpn1. This result suggests that the transgenic VWA domain fragments may interfere with the function of the full-length endogenous CPN1 protein by causing a dominant-negative effect. CPN1 was found to interact with HrBP1 (H&barbelow;arpin B&barbelow;inding P&barbelow;rotein 1&barbelow;) in a yeast two hybrid analysis (Judy Sinn, personal communication). CPN1 also pulled down HrBP1 fusion protein from total protein extracts. Fluorescence-tagged HrBP1 was mainly localized to chloroplasts; however, trace amounts of HrBP1 fusion protein were observed along the PM, especially after pathogen inoculation. This result suggests that CPN1 and HrBP1 might interact with each other after pathogen infection. Epistasis analysis suggested that the disease resistant phenotype of cpn1-1 was largely independent of salicylic acid. Taken together, our data suggest that CPN1 accumulates at cell junctions and lobes after pathogen attack, interacts with its functional associates through its VWA domain, and functions at an early step in plant defense.