Overexpression of Arabidopsis NIMIN1 results in salicylate intolerance

ABSTRACT

The transcriptional regulator NPR1 mediates salicylic acid (SA)-induced plant immunity. NPR1 is also required for tolerance to high concentrations of SA. NPR1-interacting protein, NIMIN1, represses immune response by interacting with and negating NPR1. We tested the salicylic acid tolerance of transgenic plants overexpressing NIMIN1 and found that these plants displayed SA intolerance, similar to the npr1 mutant, due to sequestration of NPR1 by NIMIN1. Plants overexpressing mutated NIMIN1 that cannot interact with NPR1 showed no SA tolerance defect. Gene expression analysis showed that NPR1 is required for SA-stress induced as well as pathogen-induced NIMIN1 expression. These results indicate that over-accumulation of a negative regulator renders plants hypersensitive to SA by limiting NPR1 function. Furthermore, NPR1 activates negative regulators such as NIMIN1 for feedback inhibition of SA signaling to maintain immune homeostasis.

Abbreviations

SA

salicylic acid

NPR1

Non-expressor of PR1

NIMIN1

NIM1 (NPR1)-interacting 1

SAR

systemic acquired resistance

Salicylic acid (SA) is a hormone produced in plants in response to biotic and abiotic stresses.¹ Infection of plants with bacterial pathogens triggers accumulation of SA as a stress signal, followed by expression of defense genes such as pathogenesis related protein 1 (PR1).² Infection or exogenous application of SA, which mimics infection, can induce defense gene expression, while triggering resistance against subsequent pathogen attack,³ a process defined as systemic acquired resistance (SAR). NPR1 is a master regulator of SAR and functions as transcriptional coactivator of defense-related genes in the nucleus, in conjunction with the bZIP family of transcription factors, namely TGA.²

In Arabidopsis, upon pathogen infection, SA is synthesized via the isochorismate synthase pathway, where chorismate into isochorismate, which is subsequently transformed into SA.⁴ The level of SA in Arabidopsis is kept in check through a feedback loop involving NPR1, which suppresses further SA accumulation.⁵ The biological importance of NPR1 in tolerating SA is evident in the case of cpr5 mutant, which displayed increased endogenous levels of SA.⁶ Chlorosis in young leaves, indicating that NPR1 is required to alleviate the toxicity caused by elevated SA levels. Similarly, young seedlings of npr1 mutant exhibited chlorosis, that was absent in wild type seedlings, when germinated on Murashige Skoog (MS) medium containing SA, a phenomenon named SA intolerance or sensitivity.⁷ These lines of evidence indicate an important role for NPR1 in SA tolerance. More recently, Zhang et al.,⁸ showed that nuclear localization of NPR1 is essential for tolerance to SA.

NIMIN1 (NIM1-Interacting; nim1 is allelic to npr1) belongs to a family of small, structurally similar proteins including NIMIN2 and NIMIN3, that were all first identified as interactors of NPR1 in a yeast 2-hybrid screen.⁹ NIMIN1 and NPR1 can also interact in planta as shown by co-immunoprecipitation in plant extracts.¹⁰ NIMIN1 associates with the C-terminal region of NPR1 through the NPR1-interaction domain, spanning amino acids 49 to 54.⁹ The binding of NIMIN1 to NPR1 is an important step in modulating the level of immune response as the nimin1 mutant displays enhanced NPR1-dependent defense gene expression.¹⁰ Specifically, NIMIN1 regulates the amplitude, rather than timing, of defense gene induction by interacting with and negating NPR1 protein.¹⁰ Like NPR1, NIMIN genes are induced by SA and the NIMIN proteins carry nuclear localization signals targeting them to the nucleus, consistent with their role as transcriptional regulators.⁹

Arabidopsis transgenics highly expressing NIMIN1 mimic the npr1 mutant by displaying reduced SA-induced PR gene expression and impairment of systemic acquired resistance.¹⁰ In contrast, plants expressing a mutant version of NIMIN1 that cannot interact with NPR1 are similar to wild type plants. These results suggest that the physical sequestration of functional NPR1 by overexpressed NIMIN1 results in an npr1-like phenotype. In order to reaffirm the functional importance of NPR1 in SA tolerance, we tested the SA tolerance phenotype of transgenic plants overexpressing myc and histidine tag-fused functional NIMIN1 (35S:NIMIN1:his:myc) and mutant nimin1-2 protein (35S:nimin1-2:his:myc); the nimin1-2 mutant protein is defective in interaction with NPR1 due to mutation of the amino acids Phenylalanine-49 (Phe-49) and Phe-50 residues in the NPR1-interaction domain to serine residues.¹⁰ As expected, 35S:NIMIN1, but not 35S:nimin1-2, replicated the SA intolerance phenotype of npr1-2 mutant (Fig. 1A), indicating that sequestration of NPR1 protein renders plants sensitive to SA-mediated stress. Immunoblot analysis was used to ascertain that these plants indeed expressed NIMIN1 fusion proteins. As seen in Fig. 1B, the myc tags associated with NIMIN1 and nimin1-2 are only detected in the transgenic plants, but not in wild type or npr1-2 mutant.

Figure 1.

SA intolerance of NIMIN1 transgenics. (A) Wild type, 35S:NIMIN1-his-myc, 35S:nimin1-2-his-myc and npr1-2 were grown on Murashige Skoog (MS) plates with (bottom left panel) or without (top left panel) supplementation with 0.4mM SA for about 2 weeks. This result was observed 4 times. (B) Immunoblot analysis of the 44 g genotypes (lower panel); transgenic NIMIN1 protein was detected using an antibody against c-myc (Cell Signaling Technology, 1:2.000). Arrow indicates the band corresponding to NIMIN1 (∼20 kDa). Asterisk represents a non-specific band, which along with the Coomassie stain (upper panel) acts a loading control. Confirmed twice.

Since NIMIN1 overexpression caused SA intolerance, we tested whether the induction of endogenous NIMIN1 contributed to SA intolerance in npr1 mutant. For this, we assayed the gene expression of NIMIN1 using quantitative PCR in seedlings grown on MS media with and without SA supplementation. NIMIN1 was induced in wild type plants by SA stress and this activation was completely dependent on NPR1, as the npr1-2 mutant lacked NIMIN1 expression (Fig. 2A). This clarifies that the SA intolerance of npr1 mutants is not due to NIMIN1 hyperinduction and that the SA intolerance of 35S:NIMIN1 is a result of physical interaction between NIMIN1 and NPR1, demonstrated before.¹⁰ To determine if the NPR1-mediated transcription of NIMIN1 was limited to seedlings displaying SA tolerance, we tested the expression of NIMIN1 in mature plants treated with SA. As observed in Fig. 2B, NPR1 was indispensible for SA-mediated expression of NIMIN1 in soil grown mature plants. Since SA mimics pathogen infection, we tested the pathogen-induced expression of NIMIN1. Indeed, not only was NIMIN1 expressed upon infection by virulent and avirulent bacterial pathogens, its induction was strikingly dependent on NPR1 (Fig. 2C). Together, these results demonstrate that while NIMIN1 inhibits NPR1 functions, including SA tolerance, through physical interaction, the native expression of NIMIN1 in wild type plants in response to SA and during immune response requires NPR1.

Figure 2.

SA- induced expression of NIMIN1 in npr1 mutant. (A) MS grown seedlings (2 wk old). Mock indicates MS plates and SA indicates supplementation of MS with 0.4 millimolar (mM) SA. (B) Mature leaves collected from 4-week old plants, 24 hours after treatment with 5 mM SA or water (mock) by root drenching for 20 minutes. (C) Leaves from 4-week old plants were collected 24 hours after infiltration with 10mM magnesium sulfate (MgSO₄) (mock), virulent bacterial pathogen, Pseudomonas syringae DC3000 (Pst) (vir) or avirulent Pst with the effector avrRpt2 (avr) suspension in MgSO₄ at a dose of 5×10⁶ colony forming units per milliliter. RNA was extracted using Trizol (Life Technologies), treated with DNAse I (Ambion), cDNA was prepared using Superscript III reverse transcriptase (Life Technologies) and quantitative RealTime PCR was performed using SYBR Green mastermix (Life Technologies). Compared to the housekeeping gene Ubiquitin 5 (UBQ5) and actin2 (ACT2) were calculated. Data was analyzed using the ΔCt method²¹ and represent the average of 3 technical replicates/2 biological replicates with standard deviation. The results were reproduced 2-4 times. For statistical analysis, One way ANOVA was performed, followed by Tukey's multiple comparison test (p<0.05). Means with the same letter are not significantly different from each other.

Plants lacking NPR1, the bZIP transcription factors TGA2,5,6 and the GRAS-family transcription factor, SCL14, all exhibit intolerance to salicylic acid or its analogs.^11,12 High concentrations of SA (>100 uM) can promote accumulation of reactive oxygen species, leading to oxidative stress.¹³ Thus, SA tolerance may be a reflection of the importance of NPR1 and the other proteins in coping with the oxidative stress caused by supraphysiological concentrations of SA. The introduction of SA intolerance by binding of NIMIN1 to NPR1 is reminiscent of a similar phenotype observed in plants expressing a truncated form of TGA2 (35S:TGA2-CT, C-terminal domain alone) that had a dominant-negative sequestration effect on NPR1 protein, resulting in toxicity on SA containing media.¹⁴ The SA intolerance of NIMIN1 overexpressing plants underscores the importance of NPR1 in SA tolerance.

The expression of NIMIN1 gene is inducible by SA¹⁰ in an NPR1-dependent manner¹⁵ and this appears to require a TGA2-binding motif, TGACG, in the promoter.¹⁶ Furthermore, NIMIN1:GUS reporter expression was abolished in plants depleted of SA, indicating a requirement of SA in NIMIN1 induction.¹⁷ From this study, it is evident that NPR1 is additionally required for the transcriptional activation of NIMIN1 during stress caused by elevated concentrations of SA as well as during pathogen infection. NIMIN1 is a bona fide negative regulator of plant immunity as NIMIN1 overexpressing plants are more susceptible and nimin1 mutant is more resistant to bacterial pathogens.¹⁰ The absolute requirement of NPR1 in the activation of negative regulators of immunity has also been observed previously, as the expression of the negative regulators, WRKY38 and WRKY62,¹⁸ and WRKY58 and TGA2 are all dependent on NPR1.^14,19,20 The NPR1-dependent activation of repressors illustrates the need for immune homeostasis during defense to prevent hyperinduction of the immune response. It is striking to note, from a previous report, that NIMIN1 inhibits its own gene expression through interaction with NPR1.¹⁰ In this study, Weigl et al.,¹⁰ reported that the expression of endogenous NIMIN1 was reduced in NIMIN1 overexpressing plants, but not the nimin1-2 lines, indicating that overexpressing NIMIN1 interacts with NPR1 in the former to prevent its endogenous gene expression.

Overall, the control of negative regulator expression by NPR1 and the physical interaction of NPR1 with the same protein appears to be part of an elegant design to modulate immune response as illustrated by the NPR1 and NIMIN1 mutants and transgenics. Since the npr1 mutant fails to mount a functional defense response, the activation of negative regulators is precluded, so there is no transcriptional activation of NIMIN1. When SA and NPR1-mediated defense is activated, NPR1 turns on NIMIN1 gene and the synthesized NIMIN1 protein (possibly after reaching a threshold) sequesters NPR1 to keep the amplitude of immune response in check; this is observed as elevated defense gene expression in nimin1 mutant. When there is excessive accumulation of NIMIN1 protein (as in NIMIN1 overexpression lines), NIMIN1 binds to NPR1 and inhibits NPR1-mediated transcriptional activation of its own gene to maintain immune homeostasis. Thus, NPR1 not only mediates feedback inhibition of SA synthesis,⁵ but also promotes feedback inhibition of SA signaling by activation of negative regulators of the SA pathway during plant immunity. Furthermore, the SA tolerance function of NPR1 also implies a possible protective role from collateral damage during immune response.

Disclosure of potential confllicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to thank Drs. Daniel Klessig and Murli Manohar at BTI, Cornell University, for kindly providing seeds and bacterial strains; Dr. Mark Sasaki at Hamilton College for kindly providing the c-myc antibody.

Funding

This work was supported by the Hamilton College Dean of Faculty and the Class of 1979 award at Hamilton College to A.C.