Salicylic Acid-binding Protein Research Articles

Simultaneous silencing of two target genes using virus-induced gene silencing technology in Nicotiana benthamiana.

Virus-induced gene silencing (VIGS) is an effective strategy for rapid gene function analysis. It is well established that the NAC transcription factor and salicylic acid (SA) signal pathway play essential roles in response to biotic stresses. However, simultaneous silencing of two target genes using VIGS in plants has been rarely reported. Therefore, in this report, we performed VIGS to silence simultaneously the SA-binding protein 2 (NbSABP2) and NbNAC1 in Nicotiana benthamiana to investigate the gene silencing efficiency of simultaneous silencing of two genes. We first cloned the full-length NbNAC1 gene, and the characterization of NbNAC1 was also analysed. Overlap extension polymerase chain reaction (PCR) analysis showed that the combination of NbSABP2 and NbNAC1 was successfully amplified. Bacteria liquid PCR confirmed that the combination of NbSABP2 and NbNAC1 was successfully inserted into the tobacco rattle virus vector. The results showed that the leaves from the NbSABP2 and NbNAC1 gene-silenced plants collapsed slightly, with browning at the base of petiole or veina. Quantitative real-time PCR results showed that the expression of NbSABP2 and NbNAC1 were significantly reduced in 12 days post silenced plants after tobacco rattle virus infiltration compared with the control plants. Overall, our results suggest that VIGS can be used to silence simultaneously two target genes.

Salicylic acid in Populus tomentosa is a remote signalling molecule induced by Botryosphaeria dothidea infection

The salicylic acid (SA) plays a critical role during the establishment of systemic acquired resistance (SAR) in uninfected plant tissues after localised exposure to a pathogen. Here, we studied SA in Populus tomentosa infected by the plant pathogen Botryosphaeria dothidea. The accumulation of SA and methyl salicylate (MeSA) occurred in chronological order in P. tomentosa. The SA and MeSA contents were greater at infected than uninfected sites. Additionally, a gene expression analysis indicated that SA might be accumulated by phenylalanine ammonialyase (PAL) and converted to MeSA by SA carboxyl methyltransferase (SAMT), while MeSA might convert to SA by SA-binding protein 2 (SABP2). The expressions of SAMT at infected sites and SABP2 at uninfected sites, respectively, were significantly up-regulated. Thus, SA might be converted to MeSA at infected sites and transported as a signalling molecule to uninfected sites, where it is converted to SA for SAR. Moreover, the expressions of pathogenesis-related genes PR-1, PR-2 and PR-5 in P. tomentosa were up-regulated by the B. dothidea infection. Our study determined that variations in SA and MeSA contents occur at infected and uninfected sites in poplar after pathogen infection and contributed to the remote signals for poplar SAR.

Transcriptional Insights into the Sugarcane-Sorghum mosaic virus Interaction

Mosaic disease is a major viral disease that severely compromises sugar content and cane production. Among mosaic disease pathogens, Sorghum mosaic virus (SrMV) is the most prevalent one. To better understand the interaction mechanism between sugarcane and SrMV, samples of SrMV-infected and virus-free leaves of sugarcane cultivar ROC22 were deep sequenced and the sequencing data was confirmed by qRT-PCR. In total, 89,338 unigenes, 481 differentially expressed unigenes and 51 homologous sequences of Potyvirus host interactor (PHI) genes were obtained. The RNA-seq data implied that, the increase of SrMV replication from endoplasmic reticulum (ER) to chloroplast led to chloroplasts damage, following the initiation of differential expression of genes related to Ca2 +, ROS, cytokinin, auxin, and ethylene signaling, and also the transcription of some defense related genes. Among 51 PHIs, the upregulation of a calmodulin-related protein gene and an ethylene-inducible transcription factor gene in two SrMV-resistant and two SrMV-susceptible sugarcane cultivars under SrMV infection suggests that these two genes could be used as potential gene knockout targets for creating SrMV incompatible germplasm. Besides, based on its inverse expression pattern in the SrMV-resistant and -susceptible sugarcane cultivars, genes encoding heat shock protein 70, chloroplastic rieske Fe/S protein, reticulon homology domain protein and salicylic acid binding protein 3 might be used as the potential markers for identifying the resistance or susceptibility of sugarcane materials to SrMV. This study should help to understand the molecular mechanisms underlying SrMV-resistance of sugarcane cultivars.

Identification and Expression Analysis of Likely Orthologs of Tobacco Salicylic Acid Binding Protein 2 in Common Beans

Identification and Expression Analysis of Likely Orthologs of Tobacco Salicylic Acid Binding Protein 2 in Common Beans

Development of transcriptome based web genomic resources of yellow mosaic disease in Vigna mungo.

Vigna mungo (Urdbean) is cultivated in the tropical and sub-tropical continental region of Asia. It is not only important source of dietary protein and nutritional elements, but also of immense value to human health due to medicinal properties. Yellow mosaic disease caused by Mungbean Yellow Mosaic India Virus is known to incur huge loss to crop, adversely affecting crop yield. Contrasting genotypes are ideal source for knowledge discovery of plant defence mechanism and associated candidate genes for varietal improvement. Whole genome sequence of this crop is yet to be completed. Moreover, genomic resources are also not freely accessible, thus available transcriptome data can be of immense use. V. mungo Transcriptome database, accessible at http://webtom.cabgrid.res.in/vmtdb/ has been developed using available data of two contrasting varieties viz., cv. VM84 (resistant) and cv. T9 (susceptible). De novo assembly was carried out using Trinity and CAP3. Out of total 240,945 unigenes, 165,894 (68.8%) showed similarity with known genes against NR database, and remaining 31.2% were found to be novel. We found 22,101 differentially expressed genes in all datasets, 44,335 putative genic SSR markers, 4105 SNPs and Indels, 64,964 transcriptional factor, 546 mature miRNA target prediction in 703 differentially expressed unigenes and 137 pathways. MAPK, salicylic acid-binding protein 2-like, pathogenesis-related protein and NBS-LRR domain were found which may play an important role in defenceagainst pathogens. This is the first web genomic resource of V. mungo for future genome annotation as well as ready to use markers for future variety improvement program.

Transcriptomic and hormonal control of boron uptake, accumulation and toxicity tolerance in poplar

Despite its low abundance in soils, Boron (B) could be highly toxic for plants in especially arid and semi-arid environments. In the current study, physiological, transcriptomic and hormonal regulations behind B toxicity response were investigated comparatively among poplar species. Previously identified clones of Populus nigra (P.n) and Populus alba (P.a) having contrasting B accumulation and leaf B toxicity symptoms were treated with elevated soil B supply in a pot trial. Physiological results of the treatment indicated better biomass growth, higher leaf chlorophyll content and more than three folds lower B accumulation in the leaves of P.a compared to P.n for all soil B supply. Microarray-based transcriptomic analysis revealed 1902 and 1006 differentially regulated transcripts for the leaves and roots of P.a under B toxicity, respectively. Several transcripts responsible for salicylic acid (SA) production (salicylic acid binding protein 2) and SA-dependent gene regulation (PR proteins, WRKYs, chitinases, proteases, lipases and protease inhibitors) were strongly upregulated specifically in P.a tissues with B toxicity. Furthermore, endogenous increase in SA content with a soil B concentration-dependent manner was measured in the roots and leaves of P.a while there was no significant alteration in concentration of the same hormone for P.n. Therefore, increase in endogenous SA concentration was strongly attributed to lower B uptake and B toxicity tolerance in P.a. In addition to SA-mediated gene regulation, genes responsible for external excretion process (mannitol dehydrogenase, UGTs, sugar transporters) were also supposed to be functional in P.a for the reduction of tissue B content under toxic conditions. On the other hand, transcriptome profiling of P.n under B toxicity revealed 1624 and 1419 altered transcripts for the leaves and roots, respectively. Several ATP binding cassette B type transporters functional in auxin transport were specifically upregulated in only P.n under B toxicity. However, endogenous auxin content was not altered in both poplar tissues in response to B toxicity. Therefore, induction of these transporter proteins in P.n without any increase in auxin content was attributed to directional transport of excess B to edge of the leaves to regulate cellular ion homeostasis in photosynthetic part of the leaves. Other P.n-specific induced transcripts such as glutathione S transferases and metallochaperones were supported this suggestion and revealed internal excretion of excess B in P.n that could be related with much higher B uptake from the roots, directional transport to the leaves and detoxification under toxic B conditions.

Abstract 2354: Identification of CDK1 as an aspirin and salicylic acid binding protein: a potential role in chemoprevention

Abstract Data emerging from the past 10 years have consolidated the rationale for investigating the use of aspirin for chemoprevention; however, the mechanisms leading to its anti-cancer effects are still being elucidated. We hypothesized that aspirin’s ability to exert chemopreventive effects may involve altering the levels and activity of cell cycle regulatory proteins. In the present study, using HT-29 colon cancer cells and other cancer cells, we demonstrated that both aspirin and its primary metabolite, salicylic acid, downregulated the protein and mRNA levels of cyclin B1 and cyclin dependent kinase-1 (CDK1). Lactacystin, a 26S proteosomal inhibitor, prevented aspirin and salicylic acid mediated degradation of cyclin B1, but not CDK1. Decrease in protein levels of cyclin B1/CDK1 was correlated with a corresponding decrease in CDK1 kinase activity. Molecular docking studies showed that aspirin and salicylic acid independently can dock on CDK1 through interactions with Leucine 83. Incubation of recombinant CDK1 with aspirin resulted in acetylation at lysine residues, this was also observed in cell culture experiments. Pre-incubation of CDK1 with salicylic acid dose dependently prevented aspirin’s ability to acetylate CDK1 in purified preparations confirming the data obtained from molecular docking studies. Our results show that CDK1 is a salicylic acid binding protein (SABP) and the chemopreventive actions of aspirin may involve modulation of levels and activity of cyclin B1 and CDK1. Citation Format: Jayarama B. Gunaje, D.Ramesh Kumar, Siddharth Kesharwani, Eduardo Callegari, Hemachand Tummala, Rakesh Dachineni. Identification of CDK1 as an aspirin and salicylic acid binding protein: a potential role in chemoprevention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2354. doi:10.1158/1538-7445.AM2017-2354

Tobacco methyl salicylate esterase mediates nonhost resistance

Nonhost resistance is a type of broad-spectrum resistance exhibited by a given plant species to most strains of a pathogen which are generally pathogenic to other plant species. In this study, we have examined the role of tobacco SABP2 (Salicylic acid-Binding Protein 2) in nonhost resistance. SABP2, a methyl salicylate esterase is a critical component of SA-signaling pathway in tobacco plants. The transgenic tobacco SABP2-silenced lines treated with tetraFA, a known inhibitor of esterase activity of SABP2 exhibited enhanced susceptibility to nonhost pathogen, Pseudomonas syringae pv. phaseolicola compared to the control plants. The increased accumulation of SABP2 transcripts upon Psp infection supports the involvement of SABP2 in nonhost resistance. The tetra-FA treated plants also showed delayed expression of pathogenesis related-1 gene upon Psp inoculations. The expression of nonhost marker genes CDM1 and HIN1 was also monitored in tobacco plants infected with host-pathogen P.s. pv. tabaci and P.s. pv. phaseolicola. Overall, results presented in this manuscript suggest that SABP2 has a role in nonhost resistance in tobacco plants.

Highlights of This Issue

The evidence that aspirin prevents cancer is compelling; however, the underlying mechanism leading to its anti-cancer effect is enigmatic. In this research article, cyclin A2 and CDK2 are demonstrated to be novel targets of aspirin and its primary metabolite, salicylic acid. Both therapeutic agents caused down-regulation of cyclin A2 and CDK2, at the protein and mRNAs levels, in a diverse panel of cancer cells and resulted in a reduction in CDK2 activity. Importantly, it is also demonstrated that CDK2 is a novel salicylic acid binding protein. These results show that the anti-cancer effects of aspirin occur through down-regulation of cell cycle regulatory proteins.Multiple myeloma resides in a hypoxic environment and shows signs of an induced hypoxic response that contributes to tumorigenesis and tumor drug resistance. Targeting this adaptive hypoxic response would have important ramifications for treating this disease. To test this, Mysore and colleagues used a Pyrrole-Imidazole polyamide that can displace a major regulator of hypoxic response, HIF1, from its DNA-binding site. Evidence shows that the polyamide suppressed growth of multiple myeloma xenografts in mice and inhibited the hypoxic response in vitro and in vivo; thus, revealing the therapeutic utility of these molecules in the treatment of multiple myeloma.Pancreatic cancer is a deadly and highly metastatic disease frequently harboring activating mutations in the KRAS oncogene. MicroRNAs (miRs) are important regulators of gene expression with an established role in cancer pathogenesis. Here, miR-31 is identified as a transcriptional target of the RAS-MAPK signaling pathway. The miR-31 host gene promoter is activated by the transcription factor ELK1. Enforced expression of miR-31 enhanced invasion-migration of pancreatic cancer cells via down regulation of the miR-31 target RASA1 and activation of RHOA. These results implicate miR-31 as a novel effector in the RAS-transformation program and demonstrate a mechanism for the metastatic behavior of pancreatic cancer.Cancer-associated fibroblasts (CAFs) are a vital cellular component of the tumor microenvironment (TME) and are a recognized source of intratumoral collagen. This study is important because it shows that CAFs not only produce collagen but also regulate the types of cross-links that form between collagen strands and contribute to the biomechanical properties of tumor stroma. Mechanistically, this activity of CAFs was mediated by lysyl hydroxylase 2 (PLOD2/LH2), which strengthens the growing body of evidence that targeting LH2 has the potential to suppress the growth and metastasis of tumors in patients.

Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes.

Although one of an enzyme's hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. It is known that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. Here we report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.

Stabilization of an α/β-Hydrolase by Introducing Proline Residues: Salicylic Acid Binding Protein 2 from Tobacco.

α/β-Hydrolases are important enzymes for biocatalysis, but their stability often limits their application. We investigated a plant esterase, salicylic acid binding protein 2 (SABP2), as a model α/β-hydrolase. SABP2 shows typical stability to urea (unfolding free energy 6.9 ± 1.5 kcal/mol) and to heat inactivation (T1/2 15min 49.2 ± 0.5 °C). Denaturation in urea occurs in two steps, but heat inactivation occurs in a single step. The first unfolding step in urea eliminates catalytic activity. Surprisingly, we found that the first unfolding likely corresponds to the unfolding of the larger catalytic domain. Replacing selected amino acid residues with proline stabilized SABP2. Proline restricts the flexibility of the unfolded protein, thereby shifting the equilibrium toward the folded conformation. Seven locations for proline substitution were chosen either by amino acid sequence alignment with a more stable homologue or by targeting flexible regions in SABP2. Introducing proline in the catalytic domain stabilized SABP2 to the first unfolding in urea for three of five cases: L46P (+0.2 M urea), S70P (+0.1), and E215P (+0.9). Introducing proline in the cap domain did not stabilize SABP2 (two of two cases), supporting the assignment that the first unfolding corresponds to the catalytic domain. Proline substitutions in both domains stabilized SABP2 to heat inactivation: L46P (ΔT1/2 15min = +6.4 °C), S70P (+5.4), S115P (+1.8), S141P (+4.9), and E215P (+4.2). Combining substitutions did not further increase the stability to urea denaturation, but dramatically increased resistance to heat inactivation: L46P−S70P ΔT1/2 15min = +25.7 °C. This straightforward proline substitution approach may also stabilize other α/β-hydrolases.

Aspirin's Active Metabolite Salicylic Acid Targets High Mobility Group Box 1 to Modulate Inflammatory Responses.

Salicylic acid (SA) and its derivatives have been used for millennia to reduce pain, fever and inflammation. In addition, prophylactic use of acetylsalicylic acid, commonly known as aspirin, reduces the risk of heart attack, stroke and certain cancers. Because aspirin is rapidly de-acetylated by esterases in human plasma, much of aspirin's bioactivity can be attributed to its primary metabolite, SA. Here we demonstrate that human high mobility group box 1 (HMGB1) is a novel SA-binding protein. SA-binding sites on HMGB1 were identified in the HMG-box domains by nuclear magnetic resonance (NMR) spectroscopic studies and confirmed by mutational analysis. Extracellular HMGB1 is a damage-associated molecular pattern molecule (DAMP), with multiple redox states. SA suppresses both the chemoattractant activity of fully reduced HMGB1 and the increased expression of proinflammatory cytokine genes and cyclooxygenase 2 (COX-2) induced by disulfide HMGB1. Natural and synthetic SA derivatives with greater potency for inhibition of HMGB1 were identified, providing proof-of-concept that new molecules with high efficacy against sterile inflammation are attainable. An HMGB1 protein mutated in one of the SA-binding sites identified by NMR chemical shift perturbation studies retained chemoattractant activity, but lost binding of and inhibition by SA and its derivatives, thereby firmly establishing that SA binding to HMGB1 directly suppresses its proinflammatory activities. Identification of HMGB1 as a pharmacological target of SA/aspirin provides new insights into the mechanisms of action of one of the world's longest and most used natural and synthetic drugs. It may also provide an explanation for the protective effects of low-dose aspirin usage.

Genetic transformation of sweet oranges to over-express SABP2 gene

Background The history of the world citrus industry is marked by a series of diseases caused by different etiologic agents. Several of them are characterized as biotrophic pathogens like Xanthomonas citri subsp. citri ,t hreeCandidatus Liberibacter spp and Citrus leprosis virus C (CiLV-C). In plant-pathogen interactions, the role of salicylic acid (SA) in activating defense related genes is well recognized [1]. The SABP2 (Salicylic acid-binding protein 2) is required to convert methyl salicylate to SA as part of the signal transduction pathway that activates systemic acquired resistance, induces PR expression, and enhances disease resistance. Due to the reduced or absence of genetic resistance to these pathogens in commercial sweet orange cultivars, the genetic transformation to over-express a gene involved in the defense response of plants is a possible alternative to produce tolerant or resistant plants to biotrophic pathogens. Methods The aim of this study was to produce ‘Hamlin’ sweet orange (Citrus sinensis L. Osb.) transgenic plants, via Agrobacterium tumefaciens, over-expressing the SABP2 gene from sweet orange driven by the constitutive promoter ubiquitin (Ubq10). The SABP2 gene was cloned into pCambia 2301 and inserted into A. tumefaciens EHA 105. The genetic transformation was performed using epicotyl segments from seedlings [2]. Results and conclusions A total of 620 explants in three independent experiments were introduced and approximately 336 shoots were regenerated. The GUS histochemical test was performed and confirmed the transformation of 30 positive shoots. These shoots are being grafted onto Carrizo citrange [Citrus sinensis x Poncirus trifoliata (L.) Raf.] seedlings grown in test tubes containing MS culture medium. The presence of the transgene will be evaluated by PCR using specifics primers that amplify part of the ubiquitin promoter and part of the gene. SABP2 expression levels in transgenic plants will be assessed through qPCR. After bud multiplication, transgenic plants will be evaluated for their response to citrus canker, HLB and leprosis.

Increasing the reaction rate of hydroxynitrile lyase from Hevea brasiliensis toward mandelonitrile by copying active site residues from an esterase that accepts aromatic esters.

The natural substrate of hydroxynitrile lyase from rubber tree (HbHNL, Hevea brasiliensis) is acetone cyanohydrin, but synthetic applications usually involve aromatic cyanohydrins such as mandelonitrile. To increase the activity of HbHNL toward this unnatural substrate, we replaced active site residues in HbHNL with the corresponding ones from esterase SABP2 (salicylic acid binding protein 2). Although this enzyme catalyzes a different reaction (hydrolysis of esters), its natural substrate (methyl salicylate) contains an aromatic ring. Three of the eleven single-amino-acid-substitution variants of HbHNL reacted more rapidly with mandelonitrile. The best was HbHNL-L121Y, with a kcat 4.2 times higher and high enantioselectivity. Site-saturation mutagenesis at position 121 identified three other improved variants. We hypothesize that the smaller active site orients the aromatic substrate more productively.

Uncovering divergent evolution of α/β-hydrolases: a surprising residue substitution needed to convert Hevea brasiliensis hydroxynitrile lyase into an esterase.

Hevea brasiliensis hydroxynitrile lyase (HbHNL) and salicylic acid binding protein 2 (SABP2, an esterase) share 45% amino acid sequence identity, the same protein fold, and even the same catalytic triad of Ser-His-Asp. However, they catalyze different reactions: cleavage of hydroxynitriles and hydrolysis of esters, respectively. To understand how other active site differences in the two enzymes enable the same catalytic triad to catalyze different reactions, we substituted amino acid residues in HbHNL with the corresponding residues from SABP2, expecting hydroxynitrile lyase activity to decrease and esterase activity to increase. Previous mechanistic studies and x-ray crystallography suggested that esterase activity requires removal of an active site lysine and threonine from the hydroxynitrile lyase. The Thr11Gly Lys236Gly substitutions in HbHNL reduced hydroxynitrile lyase activity for cleavage of mandelonitrile 100-fold, but increased esterase activity only threefold to kcat ~ 0.1 min-1 for hydrolysis of p-nitrophenyl acetate. Adding a third substitution - Glu79His - increased esterase activity more than tenfold to kcat ~ 1.6 min-1. The specificity constant (kcat/KM) for this triple substitution variant versus wild type HbHNL shifted more than one million-fold from hydroxynitrile lyase activity (acetone cyanohydrin substrate) to esterase activity (p-nitrophenyl acetate substrate). The contribution of Glu79His to esterase activity was surprising since esterases and lipases contain many different amino acids at this position, including glutamate. Saturation mutagenesis at position 79 showed that 13 of 19 possible amino acid substitutions increased esterase activity, suggesting that removal of glutamate, not addition of histidine, increased esterase activity. Molecular modeling indicates that Glu79 disrupts esterase activity in HbHNL when its negatively charged side chain distorts the orientation of the catalytic histidine. Naturally occurring glutamate at the corresponding location of Candida lipases is uncharged due to other active site differences and does not cause the same distortion. This example of the fine tuning of the same catalytic triad for different types of catalysis by subtle interactions with other active site residues shows how difficult it is to design new catalytic reactions of enzymes.

Expression of defence-related genes in avocado fruit (cv. Fuerte) infected with Colletotrichum gloeosporioides

Anthracnose is the most severe post-harvest disease of many cultivars of avocado. Identification of avocado genes involved in the defence response against Colletotrichum gloeosporioides is a crucial step towards understanding the molecular basis of resistance in avocado fruit. Following 454 sequencing and analysis of the transcriptome of Fuerte avocado fruits infected with C. gloeosporioides, quantitative real-time PCR was employed to measure the expression of some candidate resistance and defence-related genes expressed during anthracnose disease. The selected genes included those coding for the endogenous control genes (actin and glyceraldehyde 3-phosphate dehydrogenase), salicylic acid binding protein 2 (SABP2), ethylene responsive element binding protein (EREBP), leucine rich protein (LRR), catalase, endochitinase, endo-1,4-D-glucanase and pathogenesis related proteins 5 (PR 5) and 6 (PR 6). The study identified many genes involved in key components of the resistance response including recognition, signalling, transcription, the oxidative burst, PR protein induction, R genes and the hypersensitive response. Expression profiles showed that selected genes were differentially expressed after infection when compared to the uninfected sample but that there is a modulation of the defence response, suggestive of a compatible-type interaction. This transcriptome analysis provides a first elucidation of the molecular networks involved in the resistance of avocado fruit to fungal parasitic infection and provides an important contribution to functional genomic approaches to understanding resistance in non-model crop plants.

Methyl Esterase 1 (StMES1) Is Required for Systemic Acquired Resistance in Potato

Whether salicylic acid (SA) plays a role in systemic acquired resistance (SAR) signaling in potato is currently unclear because potato, unlike tobacco and Arabidopsis, contains highly elevated levels of endogenous SA. Recent studies have indicated that the SA derivative methyl salicylate (MeSA) serves as a long-distance phloem-mobile SAR signal in tobacco and Arabidopsis. Once in the distal, uninfected tissue of these plant species, MeSA must be converted into biologically active SA by the esterase activity of SA-binding protein 2 (SABP2) in tobacco or members of the AtMES family in Arabidopsis. In this study, we have identified the potato ortholog of tobacco SABP2 (StMES1) and shown that the recombinant protein converts MeSA to SA; this MeSA esterase activity is feedback inhibited by SA or its synthetic analog, 2, 2, 2, 2'-tetra-fluoroacetophenone (tetraFA). Potato plants (cv. Désirée) in which StMES1 activity was suppressed, due to either tetraFA treatment or silencing of StMES1 expression, were compromised for arachidonic acid (AA)-induced SAR development against Phytophthora infestans. Presumably due to the inability of these plants to convert MeSA to SA, the SAR-defective phenotype correlated with elevated levels of MeSA and reduced expression of pathogenesis-related (PR) genes in the untreated distal tissue. Together, these results strongly suggest that SAR signaling in potato requires StMES1, its corresponding MeSA esterase activity, and MeSA. Furthermore, the similarities between SAR signaling in potato, tobacco, and Arabidopsis suggest that at least certain SAR signaling components are conserved among plants, regardless of endogenous SA levels.

Comparative Transcriptomic Profiling of a Salt-Tolerant Wild Tomato Species and a Salt-Sensitive Tomato Cultivar

Wild halophytic tomato has long been considered as an ideal gene donor for improving salt tolerance in tomato cultivars. Extensive research has been focused on physiological and quantitative trait locus (QTL) characterization of wild tomato species in comparison with cultivated tomato. However, the global gene expression modification of wild tomato in response to salt stress is not well known. A wild tomato genotype, Solanum pimpinellifolium 'PI365967' is significantly more salt tolerant than the cultivar, Solanum lycopersicum 'Moneymaker', as evidenced by its higher survival rate and lower growth inhibition at the vegetative stage. The Affymetrix Tomato Genome Array containing 9,200 probe sets was used to compare the transcriptome of PI365967 and Moneymaker. After treatment with 200 mM NaCl for 5 h, PI365967 showed relatively fewer responsive genes compared with Moneymaker. The salt overly sensitive (SOS) pathway was found to be more active in PI365967 than in Moneymaker, coinciding with relatively less accumulation of Na(+) in shoots of PI365967. A gene encoding salicylic acid-binding protein 2 (SABP2) was induced by salinity only in PI365967, suggesting a possible role for salicylic acid signaling in the salt response of PI365967. The fact that two genes encoding lactoylglutathione lyase were salt inducible only in PI365967, together with much higher basal expression of several glutathione S-transferase genes, suggested a more effective detoxification system in PI365967. The specific down-regulation in PI365967 of a putative high-affinity nitrate transporter, known as a repressor of lateral root initiation, may explain the better root growth of this genotype during salt stress.

Featured System Salicylic Acid Binding Protein 2

Featured System Salicylic Acid Binding Protein 2

Use of a Synthetic Salicylic Acid Analog to Investigate the Roles of Methyl Salicylate and Its Esterases in Plant Disease Resistance

We previously demonstrated that salicylic acid-binding protein 2 (SABP2) of tobacco is an integral component of systemic acquired resistance (SAR). SABP2 is a methyl salicylate (MeSA) esterase that has high affinity for SA, which feedback inhibits its esterase activity. MeSA esterase activity is required in distal, healthy tissue of pathogen-infected plants to hydrolyze MeSA, which functions as a long-distance, phloem-mobile SAR signal; this hydrolysis releases the biologically active defense hormone SA. In this study, we examined the inhibitory interaction of SA with SABP2, and identified a synthetic SA analog, 2,2,2,2'-tetra-f luoroacetophenone (tetraFA) that, like SA, competitively inhibits the activity of SABP2 and targets esterases, which utilize MeSA as a substrate. However, in contrast to SA, tetraFA does not induce downstream defense responses and, therefore, is effective in planta at blocking SAR development in tobacco mosaic virus (TMV)-infected tobacco and Pseudomonas syringae-infected Arabidopsis. These results confirm the importance of SABP2 and MeSA for SAR development in tobacco and establish similar roles for MeSA and the orthologs of SABP2 in Arabidopsis. Moreover, they demonstrate that tetraFA can be used to determine whether MeSA and its corresponding esterase(s) play a role in SAR signaling in other plant species. In planta analyses using tetraFA, in conjunction with leaf detachment assays and MeSA quantification, were used to assess the kinetics with which MeSA is generated in pathogen-infected leaves, transmitted through the phloem, and processed in the distal healthy leaves. In TMV-infected tobacco, these studies revealed that critical amounts of MeSA are generated, transmitted, and processed between 48 and 72 h post primary infection.