Overexpression of (At)NPR1 in rice leads to a BTH- and environment-induced lesion-mimic/cell death phenotype

Systemic acquired resistance (SAR) is an inducible defense response that protects plants against a broad spectrum of pathogens. A central regulator of SAR in Arabidopsis is NPR1 (nonexpresser of pathogenesis-related genes). In rice, overexpression of Arabidopsis NPR1 enhances plant resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae. This report demonstrates that overexpression of (At)NPR1 in rice also triggers a lesion-mimic/cell death (LMD) phenotype. The LMD phenotype is environmentally regulated and heritable. In addition, the development of lesions and death correlates with the expression of rice defense genes and the accumulation of hydrogen peroxide. Application of the salicylic acid (SA) analog, benzo(1,2,3) thiadiazole-7-carbothioc acid S-methyl ester (BTH), potentiates this phenotype Endogenous SA levels are reduced in rice overexpressing (At)NPR1 when compared with wildtype plants, supporting the idea that (At)NPR1 may perceive and modulate the accumulation of SA. The association of (At)NPR1 expression in rice with the development of an LMD phenotype suggests that (At)NPR1 has multiple roles in plant stress responses that may affect its efficacy as a transgenic tool for engineering broad-spectrum resistance.     

Salicylic acid binds NPR3 and NPR4 to regulate NPR1-dependent defense responses

Salicylic acid (SA) is widely recognized as a key player in plant immunity. While several proteins have been previously identified as the direct targets of SA, SA-mediated plant defense signaling mechanisms remain unclear. The Nature paper from Xinnian Dong''s group demonstrates that the NPR1 paralogues NPR3 and NPR4 directly bind SA, and this binding modulates their interaction with NPR1 and thereby degradation of this key positive regulator of SA-mediated defense, shedding important new insight into the mechanism(s) of SA-mediated, NPR1-dependent plant defense signal transduction.Salicylic Acid (SA) and its derivatives (e.g., aspirin) have long been recognized for their medicinal properties as non-steroidal anti-inflammatory drugs and as pain and fever relievers. An increasing number of studies show that SA also can delay and/or prevent the development of several cancers, cardiovascular diseases, and strokes^1,2. While several SA protein targets have been identified in mammalian cells¹, their molecular and physiological modes of action remain unclear. Thus, efforts to dissect SA''s mechanisms of action continue to rely on identifying additional protein targets. Indeed, SA was recently shown to bind and activate AMP-activated protein kinase, helping to explain some of its disease-preventing effects³.SA is naturally produced in plants, and it plays diverse roles in growth, development, and responses to abiotic stresses⁴. Additionally, SA is widely recognized as a key player in multiple layers of plant disease resistance, including basal resistance, effector-triggered immunity (ETI, also termed resistance gene-mediated resistance) and systemic acquired resistance (SAR)⁵. To decipher SA-mediated plant defense signaling mechanisms, several SA-binding proteins (SABPs) have been identified, including a catalase, cytosolic ascorbate peroxidase, chloroplastic carbonic anhydrase, and methyl salicylate esterase. Extensive study of the latter protein revealed its essential role in SAR⁵. However, despite identification of the aforementioned SABPs, SA''s signaling mechanisms remain unclear. Considering SA''s many roles in plants, these SABPs may constitute only a small portion of SA''s targets; moreover, the SA receptor remained to be found.In this context, the Nature paper from Xinnian Dong''s group⁶ represents a major step forward in our understanding of SA signaling mechanisms during plant-pathogen interactions. Dong''s group has been instrumental in characterizing the function of NPR1 (Nonexpresser of Pathogenesis-Related genes 1) in plant defense⁷. While NPR1 is a key player in one of the SA-mediated defense signaling pathways, it does not appear to be an SA receptor as it does not directly bind SA⁶. Instead, SA regulates the conversion of NPR1 from an oligomeric to a monomeric form, which leads to its nuclear translocation⁸. SA also regulates NPR1 phosphorylation, which facilitates NPR1''s recruitment to a Cullin3 (CUL3) E3 ligase and subsequently proteasome-mediated degradation⁹. Now Dong''s group has demonstrated that the NPR1 paralogues NPR3 and NPR4 are adaptor proteins for the CUL3 E3 ligase that specifically target NPR1 for degradation in an SA concentration-dependent manner⁶. Supporting their conclusion, NPR3 and NPR4 contain domains typically found in CUL3 substrate adaptors, and npr3/4 single and double mutants contain elevated levels of NPR1. Furthermore, NPR3 and NPR4 directly interact with NPR1. Strikingly, SA disrupts the NPR1-NPR4 interaction, thereby making NPR1 less susceptible to degradation, whereas SA promotes the NPR1-NPR3 interaction, which makes NPR1 more accessible for degradation (Figure 1). Since NPR4 has high affinity for SA (nanomolar range) while NPR3 has low affinity for SA (micromolar range), low SA levels should reduce NPR1 degradation, whereas high SA levels should enhance it.Open in a separate windowFigure 1NPR1 homeostasis is controlled by SA binding to NPR3/NPR4 in a concentration-dependent manner. At low SA levels (High Susceptibility, left), NPR1 is unavailable to induce defense gene since it is targeted through its binding to NPR4 for degradation in proteasomes. As SA concentration increases after infection (Basal Resistance, middle), SA binds to NPR4 disrupting its interaction with NPR1. Free NPR1 can now play its role in defense gene activation. At very high concentrations (ETI, right), SA levels are sufficient to bind to NPR3 and promote its interaction with NPR1, leading to NPR1 turnover.At the biological level, nuclear accumulation of NPR1 is required for basal defense gene expression, whereas proteasome-mediated turnover is required for ETI, and a combination of NPR1 accumulation and turnover is necessary for SAR development^6,9. The results presented by Fu et al.⁶ suggest that the interplay between NPR1, NPR3/4, and an SA concentration gradient finetunes NPR1 homeostasis and thus helps specify disease resistance. According to their working model, the enhanced susceptibility exhibited by SA-deficient plants is due to unrestricted NPR4 binding to NPR1, which depletes NPR1 due to CUL3^NPR4-mediated degradation⁶. In wild-type plants, low basal SA levels may bind to NPR4, thereby allowing some NPR1 to accumulate to confer basal resistance. Following pathogen infection, recognition of pathogen effectors by plant resistance proteins induces a high level of SA in local infected tissues; in this case, CUL3^NPR3-mediated degradation would allow fast NPR1 turnover, leading to ETI. In systemic tissues, an intermediate level of SA would enable both NPR1 accumulation and turnover, leading to SAR.Clearly, the study by Fu et al.⁶ represents a major step towards elucidating the mechanism(s) of SA perception in programming defense gene expression. However, NPR3 and NPR4 may not be SA receptors in a traditional sense. An increasing body of evidence indicates the existence of SA-dependent, but NPR1-independent defense signal transduction pathways¹⁰, in which NPR3/4 may not participate. In addition, it is unknown whether NPR3/NPR4-mediated SA perception is involved in the diverse roles that this hormone plays in growth and development, or in abiotic stress. Even for NPR1-dependent defense signal transduction, it is unclear whether NPR3/NPR4 are involved in SA''s ability to induce nuclear translocation of NPR1 and/or promote NPR1 phosphorylation to facilitate the proteasome-mediated turnover. Moreover, since SA binding did not affect the gel filtration elution profile of NPR4⁶, the mechanism through which SA binding influences the ability of NPR4 (or NPR3) to bind NPR1 is currently unknown. Thus, many aspects of SA-mediated signaling remain to be explored.     

Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress

Phytochelatin (PC) plays an important role in heavy metal detoxification in plants and other living organisms. Therefore, we overexpressed an Arabidopsis PC synthase (AtPCS1) in transgenic Arabidopsis with the goal of increasing PC synthesis, metal accumulation, and metal tolerance in these plants. Transgenic Arabidopsis plants were selected, designated pcs lines, and analyzed for tolerance to cadmium (Cd). Transgenic pcs lines showed 12- to 25-fold higher accumulation of AtPCS1 mRNA, and production of PCs increased by 1.3- to 2.1-fold under 85 microM CdCl(2) stress for 3 d when compared with wild-type plants. Cd tolerance was assessed by measuring root length of plants grown on agar medium containing 50 or 85 microM CdCl(2). Pcs lines paradoxically showed hypersensitivity to Cd stress. This hypersensitivity was also observed for zinc (Zn) but not for copper (Cu). The overexpressed AtPCS1 protein itself was not responsible for Cd hypersensitivity as transgenic cad1-3 mutants overexpressing AtPCS1 to similar levels as those of pcs lines were not hypersensitive to Cd. Pcs lines were more sensitive to Cd than a PC-deficient Arabidopsis mutant, cad1-3, grown under low glutathione (GSH) levels. Cd hypersensitivity of pcs lines disappeared under increased GSH levels supplemented in the medium. Therefore, Cd hypersensitivity in pcs lines seems due to the toxicity of PCs as they existed at supraoptimal levels when compared with GSH levels.     

Overexpression of a Drosophila homolog of apolipoprotein D leads to increased stress resistance and extended lifespan

Increased Apolipoprotein D (ApoD) expression has been reported in various neurological disorders, including Alzheimer's disease, schizophrenia, and stroke, and in the aging brain . However, whether ApoD is toxic or a defense is unknown. In a screen to identify genes that protect Drosophila against acute oxidative stress, we isolated a fly homolog of ApoD, Glial Lazarillo (GLaz). In independent transgenic lines, overexpression of GLaz resulted in increased resistance to hyperoxia (100% O(2)) as well as a 29% extension of lifespan under normoxia. These flies also displayed marked improvements in climbing and walking ability after sublethal exposure to hyperoxia. Overexpression of Glaz also increased resistance to starvation without altering lipid or protein content. To determine whether GLaz might be important in protection against reperfusion injury, we subjected the flies to hypoxia, followed by recovery under normoxia. Overexpression of GLaz was protective against behavioral deficits caused in normal flies by this ischemia/reperfusion paradigm. This and the accompanying paper by Sanchez et al. (in this issue of Current Biology) are the first to manipulate the levels of an ApoD homolog in a model organism. Our data suggest that human ApoD may play a protective role and thus may constitute a therapeutic target to counteract certain neurological diseases.     

Activation of an EDS1-mediated R-gene pathway in the snc1 mutant leads to constitutive, NPR1-independent pathogen resistance

The Arabidopsis NPR1 protein is an essential regulatory component of systemic acquired resistance (SAR). Mutations in the NPR1 gene completely block the induction of SAR by signals such as salicylic acid (SA). An Arabidopsis mutant, snc1 (suppressor of npr1-1, constitutive 1), was isolated in a screen for suppressors of npr1-1. In the npr1-1 background, the snc1 mutation resulted in constitutive resistance to Pseudomonas syringae maculicola ES4326 and Peronospora parasitica Noco2. High levels of SA were detected in the mutant and shown to be required for manifestation of the snc1 phenotype. The snc1 mutation was mapped to the RPP5 resistance (R) gene cluster and the eds1 mutation that blocks RPP5-mediated resistance suppressed snc1. These data suggest that a RPP5-related resistance pathway is activated constitutively in snc1. This pathway does not employ NPR1 but requires the signal molecule SA and the function of EDS1. Moreover, in snc1, constitutive resistance is conferred in the absence of cell death, which is often associated with R-gene mediated resistance.     

Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity

Phytochelatin synthase (PCS) catalyzes the final step in the biosynthesis of phytochelatins, which are a family of cysteine-rich thiol-reactive peptides believed to play important roles in processing many thiol-reactive toxicants. A modified Arabidopsis thaliana PCS sequence (AtPCS1) was active in Escherichia coli. When AtPCS1 was overexpressed in Arabidopsis from a strong constitutive Arabidopsis actin regulatory sequence (A2), the A2::AtPCS1 plants were highly resistant to arsenic, accumulating 20-100 times more biomass on 250 and 300 microM arsenate than wild type (WT); however, they were hypersensitive to Cd(II). After exposure to cadmium and arsenic, the overall accumulation of thiol-peptides increased to 10-fold higher levels in the A2::AtPCS1 plants compared with WT, as determined by fluorescent HPLC. Whereas cadmium induced greater increases in traditional PCs (PC2, PC3, PC4), arsenic exposure resulted in the expression of many unknown thiol products. Unexpectedly, after arsenate or cadmium exposure, levels of the dipeptide substrate for PC synthesis, gamma-glutamyl cysteine (gamma-EC), were also dramatically increased. Despite these high thiol-peptide concentrations, there were no significant increases in concentrations of arsenic and cadmium in above-ground tissues in the AtPCS1 plants relative to WT plants. The potential for AtPCS1 overexpression to be useful in strategies for phytoremediating arsenic and to compound the negative effects of cadmium are discussed.     

A gain-of-function mutation in a plant disease resistance gene leads to constitutive activation of downstream signal transduction pathways in suppressor of npr1-1, constitutive 1

Plants have evolved sophisticated defense mechanisms against pathogen infections, during which resistance (R) genes play central roles in recognizing pathogens and initiating defense cascades. Most of the cloned R genes share two common domains: the central domain, which encodes a nucleotide binding adaptor shared by APAF-1, certain R proteins, and CED-4 (NB-ARC), plus a C-terminal region that encodes Leu-rich repeats (LRR). In Arabidopsis, a dominant mutant, suppressor of npr1-1, constitutive 1 (snc1), was identified previously that constitutively expresses pathogenesis-related (PR) genes and resistance against both Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2. The snc1 mutation was mapped to the RPP4 cluster. In snc1, one of the TIR-NB-LRR-type R genes contains a point mutation that results in a single amino acid change from Glu to Lys in the region between NB-ARC and LRR. Deletions of this R gene in snc1 reverted the plants to wild-type morphology and completely abolished constitutive PR gene expression and disease resistance. The constitutive activation of the defense responses was not the result of the overexpression of the R gene, because its expression level was not altered in snc1. Our data suggest that the point mutation in snc1 renders the R gene constitutively active without interaction with pathogens. To analyze signal transduction pathways downstream of snc1, epistasis analyses between snc1 and pad4-1 or eds5-3 were performed. Although the resistance signaling in snc1 was fully dependent on PAD4, it was only partially affected by blocking salicylic acid (SA) synthesis, suggesting that snc1 activates both SA-dependent and SA-independent resistance pathways.     

Negative regulation of defense responses in Arabidopsis by two NPR1 paralogs

NPR1 is required for systemic acquired resistance, and there are five NPR1 paralogs in Arabidopsis. Here we report knockout analysis of two of these, NPR3 and NPR4. npr3 single mutants have elevated basal PR-1 expression and the npr3 npr4 double mutant shows even higher expression. The double mutant plants also display enhanced resistance against virulent bacterial and oomycete pathogens. This enhanced disease resistance is partially dependent on NPR1, can be in part complemented by either wild-type NPR3 or NPR4, and is not associated with an elevated level of salicylic acid. NPR3 and NPR4 interact with TGA2, TGA3, TGA5 and TGA6 in yeast two-hybrid assays. Using bimolecular fluorescence complementation analysis, we show that NPR3 interacts with TGA2 in the nucleus of onion epidermal cells and Arabidopsis mesophyll protoplasts. Combined with our previous finding that basal PR-1 levels are also elevated in the tga2 tga5 tga6 triple mutant, we propose that NPR3 and NPR4 negatively regulate PR gene expression and pathogen resistance through their association with TGA2 and its paralogs.     

The C termini of Arabidopsis cryptochromes mediate a constitutive light response

Cryptochrome blue light photoreceptors share sequence similarity to photolyases, flavoproteins that mediate light-dependent DNA repair. However, cryptochromes lack photolyase activity and are characterized by distinguishing C-terminal domains. Here we show that the signaling mechanism of Arabidopsis cryptochrome is mediated through the C terminus. On fusion with beta-glucuronidase (GUS), both the Arabidopsis CRY1 C-terminal domain (CCT1) and the CRY2 C-terminal domain (CCT2) mediate a constitutive light response. This constitutive photomorphogenic (COP) phenotype was not observed for mutants of cct1 corresponding to previously described cry1 alleles. We propose that the C-terminal domain of Arabidopsis cryptochrome is maintained in an inactive state in the dark. Irradiation with blue light relieves this repression, presumably through an intra- or intermolecular redox reaction mediated through the flavin bound to the N-terminal photolyase-like domain.     

The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid

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Highlights? NPR1 is a salicylic acid (SA) receptor, binding specifically to SA via Cys^521/529 ? NPR1 binds copper in vivo via Cys^521/529, and metals are required for SA binding ? SA directly regulates the conformation of NPR1 by deoligomerizing NPR1 into a dimer ? The NPR1 BTB/POZ domain autoinhibits the function of the NPR1 transactivation domain