ABSTRACT
NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1; also known as NIM1) is a master regulator of systemic acquired resistance (SAR). SAR is induced by salicylic acid (SA), leading to the expression of PATHOGENESIS-RELATED (PR) genes. Current evidence suggests that NPR1 is part of a transcription complex tethered to activation sequence-1 (as-1)-like cis-acting elements in PR-1 gene promoters through TGA transcription factors, and that SA-dependent PR-1 gene expression is regulated by NIM1-INTERACTING (NIMIN) proteins. In Arabidopsis, NPR1 is active only after SA induction. Regulation of Arabidopsis NPR1 activity has been proposed to comprise cysteine-156 (Cys-156), mediating SA-induced cytoplasmic oligomer-nuclear monomer exchange, and Cys-521 and Cys-529, mediating SA-dependent transcriptional activation. Tobacco NPR1 does not harbour these residues. To understand the function of tobacco NPR1, we analysed its biochemical capabilities in a heterologous system: yeast. Tobacco NPR1 differs from Arabidopsis NPR1 in its subcellular localization and its transactivation potential. Yet, both tobacco and Arabidopsis NPR1, as well as tobacco NIM1-like1, alter some of their biochemical activities in response to SA. Whereas the addition of SA to yeast growth medium induces transcriptional activity in tobacco NPR1, its interaction with NIMIN2-type proteins is suppressed. The effects of SA are specific, sensitive and occur coordinately. They are abolished completely by mutation of the arginine residue within the invariable penta-amino acid motif LENRV, as present in the nonfunctional Arabidopsis nim1-4 allele. Furthermore, NPR1 proteins with the LENRV domain coincidently harbour a broad and strongly conserved NIMIN1/NIMIN2 binding site. Our data suggest that NPR1 and some NPR1-like proteins are sensitive to the plant hormone SA, altering some of their biochemical capabilities to enable stimulus-dependent gene expression. The sensitivity of NPR1 proteins to SA, together with their differential interaction with diverse NIMIN proteins, seems a plausible molecular basis for the timely and coordinated activation of PR genes during SAR.
Subject(s)
Arabidopsis Proteins/metabolism , Plant Proteins/metabolism , Salicylic Acid/pharmacology , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Genes, Plant , Molecular Sequence Data , Mutation , Plant Diseases/genetics , Plant Proteins/chemistry , Plant Proteins/genetics , Protein Structure, Tertiary , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino Acid , Signal Transduction , Species Specificity , Nicotiana/genetics , Nicotiana/metabolism , Trans-Activators/chemistry , Trans-Activators/genetics , Trans-Activators/metabolism , Two-Hybrid System TechniquesABSTRACT
SUMMARY: NPR1 (for Nonexpressor of PR genes; also known as NIM1) is a positive regulator of systemic acquired resistance (SAR) in Arabidopsis, which controls the induction of Pathogenesis-Related (PR) genes by salicylic acid (SA). NPR1 interacts with members of two protein families, TGA transcription factors and NIMIN (for NIM1-interacting) proteins. In Arabidopsis, NIMIN1, NIMIN2 and NIMIN3 constitute a small gene family of structurally related, yet distinct members. To unravel the biological significance of NIMIN interaction with NPR1, we searched a tobacco yeast two-hybrid cDNA library for NPR1- and NIMIN2-binding proteins. One NPR1 cDNA clone and three clones encoding NIMIN proteins were isolated. Although clearly similar to At NPR1, Nt NPR1 does not interact with At NIMIN3. Furthermore, all Nt NIMIN proteins identified are structurally related to At NIMIN2, thus forming a small NIMIN2 subfamily in tobacco. cDNA clones encoding At NIMIN1 or At NIMIN3 homologues were not identified. The function of NIMIN2 proteins was studied by expression of Nt NIMIN2a chimeric genes in tobacco. While constitutive NIMIN2a over-expression delayed PR-1 protein induction, suppression of NIMIN2 transcripts enhanced the accumulation of PR-1 proteins. In both cases, the effects of altered NIMIN2 transcript levels became evident foremost early in SAR. Notably, Nt NIMIN2 gene expression is elevated prior to the induction of the PR-1a gene. Together, the data suggest that, in tobacco, NIMIN2 proteins control PR-1 gene expression, and that NIMIN2-mediated control is exerted through transient PR-1 repression before SAR has fully developed. Furthermore, although sharing conserved domains and functions, tobacco and Arabidopsis NPR1 and NIMIN proteins are clearly distinct.
