Plants possess an innate immune system that protects them from microbial pathogens, but lack the adaptive immunity of mammals [1, 2]. Each plant cell is capable of sensing non-self entities and mounting immune responses, demonstrating remarkable functional plasticity. Recognition of pathogen-associated molecular patterns (PAMPs) results in PAMP-triggered immunity (PTI) that prevents pathogen colonization. In turn, pathogens have evolved effectors to dampen PAMP-triggered signals and thereby attenuate PTI. The plants can only activate a weak response known as basal immunity. Some host plants have evolved resistance (R) proteins to detect the presence of pathogen effectors, inducing effector-triggered immunity (ETI) . Activation of PTI or ETI leads to the generation of a blend of signal molecules, which move to distal tissues for the establishment of systemic acquired resistance (SAR). SAR is a long-lasting immunity against a broad spectrum of pathogens .
Salicylic acid (SA) is a key signal molecule for plant immunity against biotrophic and hemibiotrophic pathogens. It is not only required for the activation of SAR [5, 6], but also plays an important role in plant basal immunity and ETI –. In plants, SA can be made through two metabolic pathways, the phenylalanine ammonia lyase (PAL)-mediated phenylalanine pathway and the isochorismate synthase (ICS)-mediated isochorismate pathway. Although knockout of PAL genes significantly reduces SA production , the isochorismate pathway is thought to be more important during plant defense . Increasing cellular SA levels induces profound transcriptional changes that are largely governed by the transcription coactivator NPR1 (nonexpressor of pathogenesis-related (PR) genes). Similarly to SA, NPR1 is not only required for SAR activation, but also plays a significant role in plant basal immunity and ETI [7, 14]–. Interestingly, NPR1 is also a feedback inhibitor of SA biosynthesis. After pathogen infection, npr1 plants accumulate significantly higher levels of SA [13, 17]. Hyperaccumulation of SA causes chlorosis in juvenile leaves and inflorescences of npr1 plants . When grown on media containing high concentrations of SA, npr1 seedlings fail to develop beyond the cotyledon stage, while wild type displays tolerance to SA cytotoxicity [19, 20].
In eukaryotic cells, RNA Polymerase II (RNAPII) catalyzes the transcription of protein-encoding genes. The Elongator complex was first identified as an interactor of hyperphosphorylated RNAPII in yeast [21, 22], and subsequently purified from mammalian and plant cells [23, 24]. Elongator consists of six subunits (ELP1-6) with ELP1-3 forming the core subcomplex and ELP4-6 the accessory subcomplex [25, 26]. Among the six subunits, ELP3 is the catalytic subunit, harboring a C-terminal histone acetyltranferase (HAT) domain and an N-terminal cysteine-rich motif that resembles an iron-sulfur (Fe-S) radical S-adenosylmethionine (SAM) domain [27, 28]. ELP3 alone has intrinsic HAT activity and is capable of acetylating all four histones, whereas the six-subunit holo-Elongator predominantly acetylates lysine-14 of histone H3 and lysine-8 of histone H4 [22, 27]. Consistently, the levels of acetylated histones H3 and H4 are reduced in yeast, mammalian, and plant elp mutants [24, 27, 29]. The radical SAM domain of yeast ELP3 is a structural motif required for the integrity of the complex , whereas the archaea Methanocaldococcus jannaschii ELP3 binds and cleaves SAM, a co-substrate involved in methyl group transfers, suggesting that M. jannaschii ELP3 may have another catalytic function other than HAT activity . Indeed, a recent study indicated that the radical SAM domain of mouse ELP3, but not the HAT domain, is required for zygotic paternal genome demethylation .
Elongator is involved in diverse cellular processes including histone modification, tRNA modification, exocytosis, α-tubulin acetylation, and zygotic paternal genome demethylation [27, 32, 33]. Mutations in yeast Elongator subunits lead to resistance to the zymocin γ-toxin subunit, sensitivity to salt, caffeine and temperature [21, 34, 35]. Elongator deficiency in humans causes familial dysautonomia, an autosomal recessive disease, characterized by abnormally low numbers of neurons in the autonomic and sensory nervous systems [36, 37]. In addition, Elongator has been shown to regulate tumorigenicity and migration of melanoma cells . In plants, mutations of Elongator subunits result in pleiotropic effects including hypersensitivity to abscisic acid, resistance to oxidative stress, severely aberrant auxin phenotypes, disease susceptibility, and altered cell cycle progression [24, 39]–.
In order to identify new components in SA signaling, we performed a genetic screen for suppressors of the npr1 mutation based on restoration of SA tolerance on half-strength MS medium supplemented with 0.5 mM SA. A total of 20 gns (green npr1 seedling on SA medium) mutants showing restored SA tolerance have been isolated. We have previously described the gns1 mutant, which harbors a mutation in AtELP2 . Here we report the isolation and characterization of the gns2 mutant, in which a frameshift mutation was identified in the Arabidopsis Elongator complex subunit 3 (AtELP3). Our results indicate that, like AtELP2, AtELP3 is required for plant basal immunity and ETI but not for SAR, and demonstrate that the HAT and radical SAM domains of AtELP3 are essential for its function in plant immunity.