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.     

NPR1-dependent salicylic acid signaling inhibits Agrobacteriummediated transient expression in Arabidopsis

正Dear Editor,Arabidopsis thaliana,as a model plant,is the most well-studied plant species.One of the advantages of using Arabidopsis is that obtaining stably transformed plants using flower-dipping method is easy.However,generating transgenic plants is still time-consuming.Therefore,transient expression is frequently used to characterize protein functions.Several transient expression assays have been developed,including protoplast transfection,biolistic bombardment,and Agrobacterium-mediated transient expression.Among these assays,the Agrobacterium-mediated     

NPR1-dependent salicylic acid signaling is not involved in elevated CO2-induced heat stress tolerance in Arabidopsis thaliana

Elevated CO₂ can protect plants from heat stress (HS); however, the underlying mechanisms are largely unknown. Here, we used a set of Arabidopsis mutants such as salicylic acid (SA) signaling mutants nonexpressor of pathogenesis-related gene 1 (npr1-1 and npr1-5) and heat-shock proteins (HSPs) mutants (hsp21 and hsp70-1) to understand the requirement of SA signaling and HSPs in elevated CO₂-induced HS tolerance. Under ambient CO₂ (380 µmol mol⁻¹) conditions, HS (42°C, 24 h) drastically decreased maximum photochemical efficiency of PSII (Fv/Fm) in all studied plant groups. Enrichment of CO₂ (800 µmol mol⁻¹) with HS remarkably increased the Fv/Fm value in all plant groups except hsp70-1, indicating that NPR1-dependent SA signaling is not involved in the elevated CO₂-induced HS tolerance. These results also suggest an essentiality of HSP70-1, but not HSP21 in elevated CO₂-induced HS mitigation.     

The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance.

The cpr5 mutant was identified from a screen for constitutive expression of systemic acquired resistance (SAR). This single recessive mutation also leads to spontaneous expression of chlorotic lesions and reduced trichome development. The cpr5 plants were found to be constitutively resistant to two virulent pathogens, Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2; to have endogenous expression of the pathogenesis-related gene 1 (PR-1); and to have an elevated level of salicylic acid (SA). Lines homozygous for cpr5 and either the SA-degrading bacterial gene nahG or the SA-insensitive mutation npr1 do not express PR-1 or exhibit resistance to P. s. maculicola ES4326. Therefore, we conclude that cpr5 acts upstream of SA in inducing SAR. However, the cpr5 npr1 plants retained heightened resistance to P. parasitica Noco2 and elevated expression of the defensin gene PDF1.2, implying that NPR1-independent resistance signaling also occurs. We conclude that the cpr5 mutation leads to constitutive expression of both an NPR1-dependent and an NPR1-independent SAR pathway. Identification of this mutation indicates that these pathways are connected in early signal transduction steps and that they have overlapping functions in providing resistance.     

The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity

The Nodal and Hedgehog signaling pathways influence dorsoventral patterning at all axial levels of the CNS, but it remains largely unclear how these pathways interact to mediate patterning. Here we show that, in zebrafish, Nodal signaling is required for induction of the homeobox genes nk2.1a in the ventral diencephalon and nk2.1b in the ventral telencephalon. Hedgehog signaling is also required for telencephalic nk2.1b expression but may not be essential to establish diencephalic nk2.1a expression. Furthermore, Shh does not restore ventral diencephalic development in embryos lacking Nodal activity. In contrast, Shh does restore telencephalic nk2.1b expression in the absence of Nodal activity, suggesting that Hedgehog signaling acts downstream of Nodal activity to pattern the ventral telencephalon. Thus, the Nodal pathway regulates ventral forebrain patterning through both Hedgehog signaling-dependent and -independent mechanisms.     

Annexin1 regulates the erythroid differentiation through ERK signaling pathway

K562 cells can be used as a model of erythroid differentiation on being induced by hemin. We found that the level of annexin1 gene expression was notably increased during this indicated process. To test the hypothesis that annexin1 can regulate erythropoiesis, K562 cell clones in which annexin1 was stably increased and was knocked down by RNAi were established, respectively. With analysis by hemoglobin quantification, benzidine staining, and marker gene expression profile determination, we confirmed that hemin-induced erythroid differentiation of K562 cells was modestly stimulated by overexpression of annexin1 while it was significantly blocked by knock down of annexin1. Further studies revealed that the mechanisms of annexin1 regulation of the erythroid differentiation was partially related to the increased ERK phosphorylation and expression of p21(cip/waf), since specific inhibitor of MEK blocked the function of annexin1 in erythroid differentiation. We concluded that annexin1 exerted its erythropoiesis regulating effect by ERK pathway.     

Cyclic fluid shear stress promotes osteoblastic cells proliferation through ERK5 signaling pathway

Fluid shear stress plays an important role in bone remodeling, however, the mechanism of mechanotransduction in bone tissue remains unclear. Recently, ERK5 has been found to be involved in multiple cellular processes. This study was designed to investigate the potential involvement of ERK5 in the proliferative response of osteoblastic cells to cyclic fluid shear stress. We reported here that cyclic fluid shear stress promoted ERK5 phosphorylation in MC3T3-E1 cells. Inhibition of ERK5 phosphorylation attenuated the increased expression of AP-1 and cyclin D1 and cell proliferation induced by cyclic fluid flow, but promoted p-16 expression. Further more, we found that cyclic fluid shear stress was a better stimuli for ERK5 activation and cyclin D1 expression compared with continuous fluid shear stress. Moreover, the pharmacological ERK5 inhibitor, BIX02189, which inhibited ERK5 phosphorylation in a time-dependent manner and the suppression lasted for at least 4 h. Taken together, we demonstrate that ERK5/AP-1/cyclin D1 pathway is involved in the mechanism of osteoblasts proliferation induced by cyclic fluid shear stress, which is superior in promoting cellular proliferation compared with continuous fluid shear stress.     

AHK5 histidine kinase regulates root elongation through an ETR1-dependent abscisic acid and ethylene signaling pathway in Arabidopsis thaliana

The Arabidopsis thaliana genome encodes a small family of histidine (His) protein kinases, some of which have redundant functions as ethylene receptors, whereas others serve as cytokinin receptors. The most poorly characterized of these is authentic histidine kinase 5 (AHK5; also known as cytokinin-independent 2, CKI2). Here we characterize three independent ahk5 mutants, and show that they have a common phenotype. Our results suggest that AHK5 His-kinase acts as a negative regulator in the signaling pathway in which ethylene and ABA inhibit the root elongation through ETR1 (an ethylene receptor).