In-depth analysis of isochorismate synthase-derived metabolism in plant immunity: identification of meta-substituted benzoates and salicyloyl-malate.

Isochorismate-derived metabolism enables biosynthesis of the plant defence hormone salicylic acid (SA) and its derivatives. In Arabidopsis thaliana, the stress-induced accumulation of SA depends on ISOCHORISMATE SYNTHASE1 (ICS1), and also requires the presumed isochorismate transporter ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) and the GH3 enzyme avrPphB SUSCEPTIBLE3 (PBS3). By comparative metabolite and structural analyses, we identified several hitherto unreported ICS1- and EDS5-dependent, biotic stress-inducible Arabidopsis metabolites. These involve meta-substituted SA derivatives (5-formyl-SA, 5-carboxy-SA, 5-carboxymethyl-SA), their benzoic acid (BA) analogues (3-formyl-BA, 3-carboxy-BA, 3-carboxymethyl-BA) and, besides the previously detected salicyloyl-aspartate (SA-Asp), the ester conjugate salicyloyl-malate (SA-Mal). SA functions as a biosynthetic precursor for SA-Mal and SA-Asp, but not for the meta-substituted SA- and BA-derivatives, which accumulate to moderate levels at later stages of bacterial infection. Interestingly, Arabidopsis leaves possess oxidising activity to effectively convert meta-formyl- into meta-carboxy-SA/BAs. In contrast to SA, exogenously applied meta-substituted SA/BA-derivatives and SA-Mal exert moderate impact on plant immunity and defence-related gene expression. While the isochorismate-derived metabolites are negatively regulated by the SA receptor NON-EXPRESSOR OF PR GENES1, SA conjugates (SA-Mal, SA-Asp, SA-glucose conjugates) and meta-substituted SA/BA-derivatives are oppositely affected by PBS3. Notably, our data indicate a PBS3-independent path to isochorismate-derived SA at later stages of bacterial infection, which does not considerably impact immune-related characteristics. Moreover, our results argue against a previously proposed role of EDS5 in the biosynthesis of the immune signal N-hydroxypipecolic acid and associated transport processes. We propose a significantly extended biochemical scheme of plant isochorismate metabolism that involves an alternative generation mode for benzoate- and salicylate-derivatives.

SA and NHP glucosyltransferase UGT76B1 affects plant defense in both SID2- and NPR1-dependent and independent manner.

KEY MESSAGE: The small-molecule glucosyltransferase loss-of-function mutant ugt76b1 exhibits both SID2- or NPR1-dependent and independent facets of enhanced plant immunity, whereupon FMO1 is required for the SID2 and NPR1 independence. The small-molecule glucosyltransferase UGT76B1 inactivates salicylic acid (SA), isoleucic acid (ILA), and N-hydroxypipecolic acid (NHP). ugt76b1 loss-of-function plants manifest an enhanced defense status. Thus, we were interested how UGT76B1 genetically integrates in defense pathways and whether all impacts depend on SA and NHP. We study the integration of UGT76B1 by transcriptome analyses of ugt76b1. The comparison of transcripts altered by the loss of UGT76B1 with public transcriptome data reveals both SA-responsive, ISOCHORISMATE SYNTHASE 1/SALICYLIC ACID INDUCTION DEFICIENT 2 (ICS1/SID2)- and NON EXPRESSOR OF PR GENES 1 (NPR1)-dependent, consistent with the role of UGT76B1 in glucosylating SA, and SA-non-responsive, SID2/NPR1-independent genes. We also discovered that UGT76B1 impacts on a group of genes showing non-SA-responsiveness and regulation by infections independent from SID2/NPR1. Enhanced resistance of ugt76b1 against Pseudomonas syringae is partially independent from SID2 and NPR1. In contrast, the ugt76b1-activated resistance is completely dependent on FMO1 encoding the NHP-synthesizing FLAVIN-DEPENDENT MONOOXYGENASE 1). Moreover, FMO1 ranks top among the ugt76b1-induced SID2- and NPR1-independent pathogen responsive genes, suggesting that FMO1 determines the SID2- and NPR1-independent effect of ugt76b1. Furthermore, the genetic study revealed that FMO1, ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), SID2, and NPR1 are required for the SA-JA crosstalk and senescence development of ugt76b1, indicating that EDS1 and FMO1 have a similar effect like stress-induced SA biosynthesis (SID2) or the key SA signaling regulator NPR1. Thus, UGT76B1 influences both SID2/NPR1-dependent and independent plant immunity, and the SID2/NPR1 independence is relying on FMO1 and its product NHP, another substrate of UGT76B1.

Phosphorylation-Dependent Regulation of Guanylyl Cyclase (GC)-A and Other Membrane GC Receptors.

Receptor guanylyl cyclases (GCs) are single membrane spanning, multidomain enzymes, that synthesize cGMP in response to natriuretic peptides or other ligands. They are evolutionarily conserved from sea urchins to humans and regulate diverse physiologies. Most family members are phosphorylated on four to seven conserved serines or threonines at the beginning of their kinase homology domains. This review describes studies that demonstrate that phosphorylation and dephosphorylation are required for activation and inactivation of these enzymes, respectively. Phosphorylation sites in GC-A, GC-B, GC-E and sea urchin receptors are discussed as are mutant receptors that mimic the dephosphorylated, inactive or phosphorylated, active forms of GC-A and GC-B, respectively. A salt bridge model is described that explains why phosphorylation is required for enzyme activation. Potential kinases, phosphatases and ATP regulation of GC receptors are also discussed. Critically, knock-in mice with glutamate substitutions for receptor phosphorylation sites are described. The inability of opposing signaling pathways to inhibit cGMP synthesis in mice where GC-A or GC-B cannot be dephosphorylated demonstrates the necessity of receptor dephosphorylation in vivo. Cardiac hypertrophy, oocyte meiosis, long bone growth/achondroplasia, and bone density are regulated by GC phosphorylation, but additional processes are likely to be identified in the future.

Genome-wide analysis of NPR1-like genes in citrus species and expression analysis in response to citrus canker (Xanthomonas axonopodis pv. citri).

Citrus fruits, revered for their nutritional value, face significant threats from diseases like citrus canker, particularly impacting global citrus cultivation, notably in Pakistan. This study delves into the critical role of NPR1-like genes, the true receptors for salicylic acid (SA), in the defense mechanisms of citrus against Xanthomonas axonopodis pv. citri (Xcc). By conducting a comprehensive genome-wide analysis and phylogenetic study, the evolutionary dynamics of Citrus limon genes across diverse citrus cultivars are elucidated. Structural predictions unveil conserved domains, such as the BTB domain and ankyrin repeat domains, crucial for the defense mechanism. Motif analysis reveals essential conserved patterns, while cis-regulatory elements indicate their involvement in transcription, growth, response to phytohormones, and stress. The predominantly cytoplasmic and nuclear localization of NPR1-like genes underscores their pivotal role in conferring resistance to various citrus species. Analysis of the Ks/Ka ratio indicates a purifying selection of NPR1-like genes, emphasizing their importance in different species. Synteny and chromosomal mapping provide insights into duplication events and orthologous links among citrus species. Notably, Xac infection stimulates the expression of NPR1-like genes, revealing their responsiveness to pathogenic challenges. Interestingly, qRT-PCR profiling post-Xac infection reveals cultivar-specific alterations in expression within susceptible and resistant citrus varieties. Beyond genetic factors, physiological parameters like peroxidase, total soluble protein, and secondary metabolites respond to SA-dependent PR genes, influencing plant characteristics. Examining the impact of defense genes (NPR1) and plant characteristics on disease resistance in citrus, this study marks the inaugural investigation into the correlation between NPR1-associated genes and various plant traits in both susceptible and resistant citrus varieties to citrus bacterial canker.

Association of natriuretic peptides and receptor activity with cardio-metabolic health at middle age.

Natriuretic peptides (NP) have multiple actions benefitting cardiovascular and metabolic health. Although many of these are mediated by Guanylyl Cyclase (GC) receptors NPR1 and NPR2, their role and relative importance in vivo is unclear. The intracellular mediator of NPR1 and NPR2, cGMP, circulates in plasma and can be used to examine relationships between receptor activity and tissue responses targeted by NPs. Plasma cGMP was measured in 348 participants previously recruited in a multidisciplinary community study (CHALICE) at age 50 years at a single centre. Associations between bio-active NPs and bio-inactive aminoterminal products with cGMP, and of cGMP with tissue response, were analysed using linear regression. Mediation of associations by NPs was assessed by Causal Mediation Analysis (CMA). ANP's contribution to cGMP far exceed those of other NPs. Modelling across three components (demographics, NPs and cardiovascular function) shows that ANP and CNP are independent and positive predictors of cGMP. Counter intuitively, findings from CMA imply that in specific tissues, NPR1 responds more to BNP stimulation than ANP. Collectively these findings align with longer tissue half-life of BNP, and direct further therapeutic interventions towards extending tissue activity of ANP and CNP.

Defense signaling pathways in resistance to plant viruses: Crosstalk and finger pointing.

Resistance to infection by plant viruses involves proteins encoded by plant resistance (R) genes, viz., nucleotide-binding leucine-rich repeats (NLRs), immune receptors. These sensor NLRs are activated either directly or indirectly by viral protein effectors, in effector-triggered immunity, leading to induction of defense signaling pathways, resulting in the synthesis of numerous downstream plant effector molecules that inhibit different stages of the infection cycle, as well as the induction of cell death responses mediated by helper NLRs. Early events in this process involve recognition of the activation of the R gene response by various chaperones and the transport of these complexes to the sites of subsequent events. These events include activation of several kinase cascade pathways, and the syntheses of two master transcriptional regulators, EDS1 and NPR1, as well as the phytohormones salicylic acid, jasmonic acid, and ethylene. The phytohormones, which transit from a primed, resting states to active states, regulate the remainder of the defense signaling pathways, both directly and by crosstalk with each other. This regulation results in the turnover of various suppressors of downstream events and the synthesis of various transcription factors that cooperate and/or compete to induce or suppress transcription of either other regulatory proteins, or plant effector molecules. This network of interactions results in the production of defense effectors acting alone or together with cell death in the infected region, with or without the further activation of non-specific, long-distance resistance. Here, we review the current state of knowledge regarding these processes and the components of the local responses, their interactions, regulation, and crosstalk.

Enhancement of broad-spectrum disease resistance in wheat through key genes involved in systemic acquired resistance.

Systemic acquired resistance (SAR) is an inducible disease resistance phenomenon in plant species, providing plants with broad-spectrum resistance to secondary pathogen infections beyond the initial infection site. In Arabidopsis, SAR can be triggered by direct pathogen infection or treatment with the phytohormone salicylic acid (SA), as well as its analogues 2,6-dichloroisonicotinic acid (INA) and benzothiadiazole (BTH). The SA receptor non-expressor of pathogenesis-related protein gene 1 (NPR1) protein serves as a key regulator in controlling SAR signaling transduction. Similarly, in common wheat (Triticum aestivum), pathogen infection or treatment with the SA analogue BTH can induce broad-spectrum resistance to powdery mildew, leaf rust, Fusarium head blight, and other diseases. However, unlike SAR in the model plant Arabidopsis or rice, SAR-like responses in wheat exhibit unique features and regulatory pathways. The acquired resistance (AR) induced by the model pathogen Pseudomonas syringae pv. tomato strain DC3000 is regulated by NPR1, but its effects are limited to the adjacent region of the same leaf and not systemic. On the other hand, the systemic immunity (SI) triggered by Xanthomonas translucens pv. cerealis (Xtc) or Pseudomonas syringae pv. japonica (Psj) is not controlled by NPR1 or SA, but rather closely associated with jasmonate (JA), abscisic acid (ABA), and several transcription factors. Furthermore, the BTH-induced resistance (BIR) partially depends on NPR1 activation, leading to a broader and stronger plant defense response. This paper provides a systematic review of the research progress on SAR in wheat, emphasizes the key regulatory role of NPR1 in wheat SAR, and summarizes the potential of pathogenesis-related protein (PR) genes in genetically modifying wheat to enhance broad-spectrum disease resistance. This review lays an important foundation for further analyzing the molecular mechanism of SAR and genetically improving broad-spectrum disease resistance in wheat.

Genetic Variants Associated with Hypertension Risk: Progress and Implications.

Background: Genetic variants causing diseases with hypertension as a secondary feature have previously been identified. Studies focussing on primary hypertension have utilised common and latterly rare genetic variants in attempts to elucidate the genetic contribution to the risk of primary hypertension. Summary: Using genome-wide association studies (GWASs), associations of hypertension with hundreds of common genetic variants have been reported, implicating thousands of genes. Individual variants have small effect sizes and cumulatively account for around 6% of genetic risk. The common variant signal is enriched for relevant tissues and physiological processes, while some variants are associated with traits expected to have secondary impacts on hypertension risk, such as fruit intake, BMI, or time watching television. Studies using rare variants obtained from exome sequence data have implicated a small number of genes for which impaired function has moderate effects on blood pressure and/or hypertension risk. Notably, genetic variants which impair elements of guanylate cyclase activation, stimulated by either natriuretic hormones or nitric oxide, increase hypertension risk. Conversely, variants impairing dopamine beta-hydroxylase or renin production are associated with lower blood pressure. Variants for which a definite effect can be designated remain cumulatively extremely rare and again make only a small contribution to overall genetic risk. Although these results are of interest, it is not clear that they provide radical new insights or identify drug targets which were not previously known. Nor does it seem that genetic testing could be useful in terms of quantifying disease risk or guiding treatment. Key Messages: Research has increased our knowledge about the relationship between naturally occurring genetic variation and risk of hypertension. Although some results serve to confirm our understanding of underlying physiology, their value in terms of potentially leading to practical advances in the management of hypertension appears questionable.

The tRNA thiolation-mediated translational control is essential for plant immunity.

Plants have evolved sophisticated mechanisms to regulate gene expression to activate immune responses against pathogen infections. However, how the translation system contributes to plant immunity is largely unknown. The evolutionarily conserved thiolation modification of transfer RNA (tRNA) ensures efficient decoding during translation. Here, we show that tRNA thiolation is required for plant immunity in Arabidopsis. We identify a cgb mutant that is hyper-susceptible to the pathogen Pseudomonas syringae. CGB encodes ROL5, a homolog of yeast NCS6 required for tRNA thiolation. ROL5 physically interacts with CTU2, a homolog of yeast NCS2. Mutations in either ROL5 or CTU2 result in loss of tRNA thiolation. Further analyses reveal that both transcriptome and proteome reprogramming during immune responses are compromised in cgb. Notably, the translation of salicylic acid receptor NPR1 is reduced in cgb, resulting in compromised salicylic acid signaling. Our study not only reveals a regulatory mechanism for plant immunity but also uncovers an additional biological function of tRNA thiolation.

A fungal CFEM-containing effector targets NPR1 regulator NIMIN2 to suppress plant immunity.

Colletotrichum fructicola causes a broad range of plant diseases worldwide and secretes many candidate proteinous effectors during infection, but it remains largely unknown regarding their effects in conquering plant immunity. Here, we characterized a novel effector CfEC12 that is required for the virulence of C. fructicola. CfEC12 contains a CFEM domain and is highly expressed during the early stage of host infection. Overexpression of CfEC12 suppressed BAX-triggered cell death, callose deposition and ROS burst in Nicotiana benthamiana. CfEC12 interacted with apple MdNIMIN2, a NIM1-interacting (NIMIN) protein that putatively modulates NPR1 activity in response to SA signal. Transient expression and transgenic analyses showed that MdNIMIN2 was required for apple resistance to C. fructicola infection and rescued the defence reduction in NbNIMIN2-silenced N. benthamiana, supporting a positive role in plant immunity. CfEC12 and MdNPR1 interacted with a common region of MdNIMIN2, indicating that CfEC12 suppresses the interaction between MdNIMIN2 and MdNPR1 by competitive target binding. In sum, we identified a fungal effector that targets the plant salicylic acid defence pathway to promote fungal infection.

NPR1, a key immune regulator for plant survival under biotic and abiotic stresses.

Nonexpressor of pathogenesis-related genes 1 (NPR1) was discovered in Arabidopsis as an activator of salicylic acid (SA)-mediated immune responses nearly 30 years ago. How NPR1 confers resistance against a variety of pathogens and stresses has been extensively studied; however, only in recent years have the underlying molecular mechanisms been uncovered, particularly NPR1's role in SA-mediated transcriptional reprogramming, stress protein homeostasis, and cell survival. Structural analyses ultimately defined NPR1 and its paralogs as SA receptors. The SA-bound NPR1 dimer induces transcription by bridging two TGA transcription factor dimers, forming an enhanceosome. Moreover, NPR1 orchestrates its multiple functions through the formation of distinct nuclear and cytoplasmic biomolecular condensates. Furthermore, NPR1 plays a central role in plant health by regulating the crosstalk between SA and other defense and growth hormones. In this review, we focus on these recent advances and discuss how NPR1 can be utilized to engineer resistance against biotic and abiotic stresses.

Analysis of Rare Variants in 470,000 Exome-Sequenced UK Biobank Participants Implicates Novel Genes Affecting Risk of Hypertension.

Introduction: A previous study of 200,000 exome-sequenced UK Biobank participants to test for association of rare coding variants with hypertension implicated two genes at exome-wide significance, DNMT3A and FES. A total of 42 genes had an uncorrected p value <0.001. These results were followed up in a larger sample of 470,000 exome-sequenced participants. Methods: Weighted burden analysis of rare coding variants in a new sample of 97,050 cases and 172,263 controls was carried out for these 42 genes. Those showing evidence for association were then analysed in the combined sample of 167,127 cases and 302,691 controls. Results: The association of DNMT3A and FES with hypertension was replicated in the new sample and they and the previously implicated gene NPR1, which codes for a membrane-bound guanylate cyclase, were all exome-wide significant in the combined sample. Also exome-wide significant as risk genes for hypertension were GUCY1A1, ASXL1, and SMAD6, while GUCY1B1 had a nominal p value of <0.0001. GUCY1A1 and GUCY1B1 code for subunits of a soluble guanylate cyclase. For two genes, DBH, which codes for dopamine beta hydroxylase, and INPPL1, rare coding variants predicted to impair gene function were protective against hypertension, again with exome-wide significance. Conclusion: The findings offer new insights into biological risk factors for hypertension which could be the subject of further investigation. In particular, genetic variants predicted to impair the function of either membrane-bound guanylate cyclase, activated by natriuretic peptides, or soluble guanylate cyclase, activated by nitric oxide, increase risk of hypertension. Conversely, variants impairing the function of dopamine beta hydroxylase, responsible for the synthesis of norepinephrine, reduce hypertension risk.

The expression of the NPR1-dependent defense response pathway genes in Persea americana (Mill.) following infection with Phytophthora cinnamomi.

A plant's defense against pathogens involves an extensive set of phytohormone regulated defense signaling pathways. The salicylic acid (SA)-signaling pathway is one of the most well-studied in plant defense. The bulk of SA-related defense gene expression and the subsequent establishment of systemic acquired resistance (SAR) is dependent on the nonexpressor of pathogenesis-related genes 1 (NPR1). Therefore, understanding the NPR1 pathway and all its associations has the potential to provide valuable insights into defense against pathogens. The causal agent of Phytophthora root rot (PRR), Phytophthora cinnamomi, is of particular importance to the avocado (Persea americana) industry, which encounters considerable economic losses on account of this pathogen each year. Furthermore, P. cinnamomi is a hemibiotrophic pathogen, suggesting that the SA-signaling pathway plays an essential role in the initial defense response. Therefore, the NPR1 pathway which regulates downstream SA-induced gene expression would be instrumental in defense against P. cinnamomi. Thus, we identified 92 NPR1 pathway-associated orthologs from the P. americana West Indian pure accession genome and interrogated their expression following P. cinnamomi inoculation, using RNA-sequencing data. In total, 64 and 51 NPR1 pathway-associated genes were temporally regulated in the partially resistant (Dusa®) and susceptible (R0.12) P. americana rootstocks, respectively. Furthermore, 42 NPR1 pathway-associated genes were differentially regulated when comparing Dusa® to R0.12. Although this study suggests that SAR was established successfully in both rootstocks, the evidence presented indicated that Dusa® suppressed SA-signaling more effectively following the induction of SAR. Additionally, contrary to Dusa®, data from R0.12 suggested a substantial lack of SA- and NPR1-related defense gene expression during some of the earliest time-points following P. cinnamomi inoculation. This study represents the most comprehensive investigation of the SA-induced, NPR1-dependent pathway in P. americana to date. Lastly, this work provides novel insights into the likely mechanisms governing P. cinnamomi resistance in P. americana.

High-Density Chitosan Induces a Biochemical and Molecular Response in Coffea arabica during Infection with Hemileia vastatrix.

The coffee industry faces coffee leaf rust caused by Hemileia vastratix, which is considered the most devastating disease of the crop, as it reduces the photosynthetic rate and limits productivity. The use of plant resistance inducers, such as chitosan, is an alternative for the control of the disease by inducing the synthesis of phytoalexins, as well as the activation of resistance genes. Previously, the effect of chitosan from different sources and physicochemical properties was studied; however, its mechanisms of action have not been fully elucidated. In this work, the ability of food-grade high-density chitosan (0.01% and 0.05%) to control the infection caused by the pathogen was evaluated. Subsequently, the effect of high-density chitosan (0.05%) on the induction of pathogenesis-related gene expression (GLUC, POX, PAL, NPR1, and CAT), the enzymatic activity of pathogenesis-related proteins (GLUC, POX, SOD, PPO, and APX), and phytoalexin production were evaluated. The results showed that 0.05% chitosan increased the activity and gene expression of ß-1,3 glucanases and induced a differentiated response in enzymes related to the antioxidant system of plants. In addition, a correlation was observed between the activities of polyphenol oxidase and the production of phytoalexin, which allowed an effective defense response in coffee plants.

Quercetin induces pathogen resistance through the increase of salicylic acid biosynthesis in Arabidopsis.

Quercetin is a flavonol belonging to the flavonoid group of polyphenols. Quercetin is reported to have a variety of biological functions, including antioxidant, pigment, auxin transport inhibitor and root nodulation factor. Additionally, quercetin is known to be involved in bacterial pathogen resistance in Arabidopsis through the transcriptional increase of pathogenesis-related (PR) genes. However, the molecular mechanisms underlying how quercetin promotes pathogen resistance remain elusive. In this study, we showed that the transcriptional increases of PR genes were achieved by the monomerization and nuclear translocation of nonexpressor of pathogenesis-related proteins 1 (NPR1). Interestingly, salicylic acid (SA) was approximately 2-fold accumulated by the treatment with quercetin. Furthermore, we showed that the increase of SA biosynthesis by quercetin was induced by the transcriptional increases of typical SA biosynthesis-related genes. In conclusion, this study strongly suggests that quercetin induces bacterial pathogen resistance through the increase of SA biosynthesis in Arabidopsis.

Reactive Oxygen Species in Drought-Induced Stomatal Closure: The Potential Roles of NPR1.

Stomatal closure is a vital, adaptive mechanism that plants utilize to minimize water loss and withstand drought conditions. We will briefly review the pathway triggered by drought that governs stomatal closure, with specific focuses on salicylic acid (SA) and reactive oxygen species (ROS). We propose that the non-expressor of PR Gene 1 (NPR1), a protein that protects plants during pathogen infections, also responds to SA during drought to sustain ROS levels and prevent ROS-induced cell death. We will examine the evidence underpinning this hypothesis and discuss potential strategies for its practical implementation.

Salicylic acid accelerates carbon starvation-induced leaf senescence in Arabidopsis thaliana by inhibiting autophagy through Nonexpressor of pathogenesis-related genes 1.

In plants, leaf senescence is regulated by several factors, including age and carbon starvation. The molecular mechanism of age-regulated developmental leaf senescence differs from that of carbon starvation-induced senescence. Salicylic acid (SA) and Nonexpressor of pathogenesis-related genes 1 (NPR1) play important roles in promoting developmental leaf senescence. However, the relationship between SA signaling and carbon starvation-induced leaf senescence is not currently well understood. Here, we used Arabidopsis thaliana as material and found that carbon starvation-induced leaf senescence was accelerated in the SA dihydroxylase mutants s3hs5h compared to the Columbia ecotype (Col). Exogenous SA treatment significantly promoted carbon starvation-induced leaf senescence, especially in NPR1-GFP. Increasing the endogenous SA and overexpression of NPR1 inhibited carbon starvation-induced autophagy. However, mutation of NPR1 delayed carbon starvation-induced leaf senescence, increased autophagosome production and accelerated autophagic degradation of the Neighbor of BRCA1 gene 1 (NBR1). In conclusion, SA promotes carbon starvation-induced leaf senescence by inhibiting autophagy via NPR1.

High air humidity dampens salicylic acid pathway and NPR1 function to promote plant disease.

The occurrence of plant disease is determined by interactions among host, pathogen, and environment. Air humidity shapes various aspects of plant physiology and high humidity has long been known to promote numerous phyllosphere diseases. However, the molecular basis of how high humidity interferes with plant immunity to favor disease has remained elusive. Here we show that high humidity is associated with an "immuno-compromised" status in Arabidopsis plants. Furthermore, accumulation and signaling of salicylic acid (SA), an important defense hormone, are significantly inhibited under high humidity. NPR1, an SA receptor and central transcriptional co-activator of SA-responsive genes, is less ubiquitinated and displays a lower promoter binding affinity under high humidity. The cellular ubiquitination machinery, particularly the Cullin 3-based E3 ubiquitin ligase mediating NPR1 protein ubiquitination, is downregulated under high humidity. Importantly, under low humidity the Cullin 3a/b mutant plants phenocopy the low SA gene expression and disease susceptibility that is normally observed under high humidity. Our study uncovers a mechanism by which high humidity dampens a major plant defense pathway and provides new insights into the long-observed air humidity influence on diseases.

Inhibition of NPR1 Leads to Shoot Growth Improvement under Low-Calcium Conditions in Arabidopsis.

Under low-Ca conditions, plants accumulate salicylic acid (SA) and induce SA-responsive genes. However, the relationship between SA and low-Ca tolerance remains unclear. Here, we demonstrated that the inhibition or suppression of nonexpressor of pathogenesis-related 1 (NPR1) activity, a major regulator of the SA signaling pathway in the defense response, improves shoot growth under low-Ca conditions. Furthermore, mutations in phytoalexin-deficient 4 (PAD4) or enhanced disease susceptibility 1 (EDS1), which are upstream regulators of NPR1, improved shoot growth under low-Ca conditions, suggesting that NPR1 suppressed growth under low-Ca conditions. In contrast, growth of SA induction-deficient 2-2 (sid2-2), which is an SA-deficient mutant, was sensitive to low Ca levels, suggesting that SA accumulation by SID2 was not related to growth inhibition under low-Ca conditions. Additionally, npr1-1 showed low-Ca tolerance, and the application of tenoxicam-an inhibitor of the NPR1-mediated activation of gene expression-also improved shoot growth under low Ca conditions. The low-Ca tolerance of double mutants pad4-1, npr1-1 and eds1-22 npr1-1 was similar to that of the single mutants, suggesting that PAD4 and EDS1 are involved in the same genetic pathway in suppressing growth under low-Ca conditions as NPR1. Cell death and low-Ca tolerance did not correlate among the mutants, suggesting that growth improvement in the mutants was not due to cell death inhibition. In conclusion, we revealed that NPR1 suppresses plant growth under low-Ca conditions and that the other SA-related genes influence plant growth and cell death.

Salicylic acid regulates phenolic acid biosynthesis via SmNPR1-SmTGA2/SmNPR4 modules in Salvia miltiorrhiza.

Phenolic acids are the main active ingredients in Salvia miltiorrhiza, which can be used for the treatment of many diseases, particularly cardiovascular diseases. It is known that salicylic acid (SA) can enhance phenolic acid content, but the molecular mechanism of its regulation is still unclear. Nonexpresser of PR genes 1 (NPR1) plays a positive role in the SA signaling pathway. In this study, we identified a SmNPR1 gene that responds to SA induction and systematically investigated its function. We found that SmNPR1 positively affected phenolic acid biosynthesis. Then, we identified a novel TGA transcription factor, SmTGA2, which interacts with SmNPR1. SmTGA2 positively regulates phenolic acid biosynthesis by directly up-regulating SmCYP98A14 expression. After double-gene transgenic analysis and other biochemical assays, it was found that SmNPR1 and SmTGA2 work synergistically to regulate phenolic acid biosynthesis. In addition, SmNPR4 forms a heterodimer with SmNPR1 to inhibit the function of SmNPR1, and SA can alleviate this effect. Collectively, these findings elucidate the molecular mechanism underlying the regulation of phenolic acid biosynthesis by SmNPR1-SmTGA2/SmNPR4 modules and provide novel insights into the SA signaling pathway regulating plant secondary metabolism.