The Arabidopsis thaliana NONEXPRESSER OF PR GENES1 (NPR1, also known as NIM1) protein is an essential positive regulator of salicylic acid (SA)-induced PATHOGENESIS-RELATED (PR) gene expression and systemic acquired resistance (SAR). PR gene activity is regulated at the level of redox-dependent nuclear transport of NPR1. NPR1 interacts with members of the TGA family of transcription factors that are known to bind to SA-responsive elements in the PR-1 promoter.In an attempt to identify proteins involved in SA-mediated signal transduction, we previously described the isolation of three novel genes encoding distinct albeit structurally related proteins designated NIMIN1 (for NIM1-INTERACTING1), NIMIN2, and NIMIN3 that interact with NPR1 in the yeast two-hybrid system. Here, we show that NIMIN1 and NPR1 can be copurified from plant extracts, providing biochemical evidence for their interaction. We provide functional evidence for this interaction by describing transgenic plants constitutively expressing high amounts of NIMIN1. These plants show reduced SA-mediated PR gene induction and a compromised SAR, thus mimicking the described phenotype conferred by npr1. Moreover, they showed reduced RESISTANCE gene-mediated protection. These effects were dependent on the ability of NIMIN1 to interact with NPR1. Mutant plants with a T-DNA insertion in NIMIN1 as well as transgenic plants with reduced NIMIN1 mRNA levels showed hyperactivation of PR-1 gene expression after SA treatment but no effect on the disease resistance phenotype. Our results strongly suggest that NIMIN1 negatively regulates distinct functions of NPR1, providing a mechanism to modulate specific features of SAR.

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.

NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR) in Arabidopsis. Using the yeast two-hybrid system, we have identified three novel genes, NIMIN-1, NIMIN-2 and NIMIN-3 (NIMIN for NIM1-interacting) that encode structurally related proteins interacting physically with NPR1/NIM1. NIMIN-1 and NIMIN-2 both bind strongly to NPR1/NIM1 via a common binding motif interacting with the C-terminal moiety of NPR1/NIM1, whereas NIMIN-3 interacts with NPR1/NIM1 via the N-terminal part of NPR1/NIM1. In addition, NIMIN-1, NIMIN-2, and NIMIN-3 are able to interact via NPR1/NIM1 with basic leucine zipper transcription factors of the TGA family in a yeast tri-hybrid system. A mutant protein of NPR1/NIM1, npr1-2, which has been shown to be severely impaired in induction of SAR gene expression, failed to bind the NIMIN proteins. The NIMIN genes are expressed in Arabidopsis plants in response to SAR-inducing treatments, and the NIMIN proteins, like NPR1/NIM1, carry functional nuclear localization signals as revealed by expression of fusion proteins in yeast and in transgenic plants. Taken together, these data indicate that the NIMIN proteins, via physical interaction with NPR1/NIM1, are part of the signal transduction pathway leading to SAR gene expression in Arabidopsis.

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.

Tobacco pathogenesis‐related protein 1a (PR‐1a) is induced in plants during the hypersensitive response (HR) after exposure of plants to salicylic acid (SA) and by developmental cues. Gene activation by these diverse stimuli is mediated via an as‐1‐like element in the PR‐1a upstream region. To further analyze the significance of this cis‐acting sequence, an authentic as‐1 element from the cauliflower mosaic virus 35S RNA promoter was inserted into the PR‐1a promoter in place of the as‐1‐like motif. Reporter gene analysis in transgenic tobacco plants demonstrated that as‐1 can functionally replace the as‐1‐like element in the PR‐1a promoter in response to all stimuli. However, reporter gene induction from the as‐1 carrying promoter was enhanced in response to SA compared to the wild‐type promoter, and the ratio of reporter gene activities in SA treated leaf tissue to tissue exhibiting the HR increased with the as‐1 promoter construct. Our findings support a model where PR‐1a gene expression relies on at least two distinct signal transduction pathways initiated by SA and by a yet unknown signal produced during the HR, that promote different, albeit related, transcription complexes on the PR‐1a as‐1‐like element. Analysis of PR‐1 proteins in plants expressing salicylate hydroxylase yielded additional evidence that an HR dependent pathway leads to high level PR‐1 gene induction in tobacco.