Identification of regions of the tomato gamma-glutamyl kinase that are involved in allosteric regulation by proline

The first step of proline biosynthesis is catalyzed by gamma-glutamyl kinase (GK). To better understand the feedback inhibition properties of GK, we randomly mutagenized a plasmid carrying tomato tomPRO1 cDNA, which encodes proline-sensitive GK. A pool of mutagenized plasmids was transformed into an Escherichia coli GK mutant, and proline-overproducing derivatives were selected on minimal medium containing the toxic proline analog 3,4-dehydro-dl-proline. Thirty-two mutations that conferred 3,4-dehydro-dl-proline resistance were obtained. Thirteen different single amino acid substitutions were identified at nine different residues. The residues were distributed throughout the N-terminal two-thirds of the polypeptide, but 9 mutations affecting 6 residues were in a cluster of 16 residues. GK assays revealed that these amino acid substitutions caused varying degrees of diminished sensitivity to proline feedback inhibition and also resulted in a range of increased proline accumulation in vivo. GK belongs to a family of amino acid kinases, and a predicted three-dimensional model of this enzyme was constructed on the basis of the crystal structures of three related kinases. In the model, residues that were identified as important for allosteric control were located close to each other, suggesting that they may contribute to the structure of a proline binding site. The putative allosteric binding site partially overlaps the dimerization and substrate binding domains, suggesting that the allosteric regulation of GK may involve a direct structural interaction between the proline binding site and the dimerization and catalytic domains.     

Mitogen-activated protein kinase (MAPK) phosphatase 3-mediated cross-talk between MAPKs ERK2 and p38alpha

MAPK phosphatase 3 (MKP3) is highly specific for ERK1/2 inactivation via dephosphorylation of both phosphotyrosine and phosphothreonine critical for enzymatic activation. Here, we show that MKP3 is able to effectively dephosphorylate the phosphotyrosine, but not phosphothreonine, in the activation loop of p38α in vitro and in intact cells. The catalytic constant of the MKP3 reaction for p38α is comparable with that for ERK2. Remarkably, MKP3, ERK2, and phosphorylated p38α can form a stable ternary complex in solution, and the phosphatase activity of MKP3 toward p38α substrate is allosterically regulated by ERK2-MKP3 interaction. This suggests that MKP3 not only controls the activities of ERK2 and p38α but also mediates cross-talk between these two MAPK pathways. The crystal structure of bisphosphorylated p38α has been determined at 2.1 Å resolution. Comparisons between the phosphorylated MAPK structures reveal the molecular basis of MKP3 substrate specificity.     

Identification of growth processes involved in QTLs for tomato fruit size and composition

Many quantitative trait loci (QTLs) for quality traits havebeen located on the tomato genetic map, but introgression offavourable wild alleles into large fruited species is hamperedby co-localizations of QTLs with antagonist effects. The aimof this study was to assess the growth processes controlledby the main QTLs for fruit size and composition. Four nearlyisogenic lines (NILs) derived from an intraspecific cross betweena tasty cherry tomato (Cervil) and a normal-tasting large fruittomato (Levovil) were studied. The lines carried one (L2, L4,and L9) or five (Lx) introgressions from Cervil on chromosomes1, 2, 4, and 9. QTLs for fruit size could be mainly associatedwith cell division processes in L2 and L9, whereas cell expansionwas rather homogeneous among the genotypes, except Cervil forwhich the low expansion rate was attributed to low cell plasticity.The link between endoreduplication and fruit size remained unclear,as cell or fruit sizes were positively correlated with the cellDNA content, but not with the endoreduplication factor. QTLsfor fruit composition reflected differences in water accumulationrather than in sugar accumulation, except in L9 for which theup-regulation of sucrose unloading and hexose transport and/orstarch synthesis was suggested. This may explain the increasedamount of carbon allocated to cell structures in L9, which couldbe related to a QTL for fruit texture. In Lx, these effectswere attenuated, except on fruit size and cell division. Finally,the region on top of chromosome 9 may control size and compositionattributes in tomato, by a combination of QTL effects on celldivision, cell wall synthesis, and carbon import and metabolism. Key words: Cell division and expansion, endoreduplication, fruit quality, near isogenic line, osmotic regulation, quantitative trait locus, Solanum lycopersicum, starch, sugar and acid contents Received 22 July 2008; Revised 17 October 2008 Accepted 20 October 2008     

Identification of genes involved in mucosal defense and inflammation associated with normal enteric bacteria

Normal luminal bacteria and their products play a role in experimental colitis and inflammatory bowel disease. However, what molecules from what cells are responsible for mounting and maintaining the mucosal defense against luminal flora is still uncertain. The aim of this study was to identify epithelial gene products involved in mucosal defense and inflammation associated with ubiquitous enteric bacteria. Germ-free ICR mice were given an oral bacterial suspension prepared from conventional components (bacterial reconstitution). Small intestinal and colonic epithelial cells were isolated from bacteria-reconstituted, germ-free, and specific pathogen-free mice. Differential gene expression was investigated by differential display, Northern blot, and sequence analysis. Bacterial reconstitution resulted in acute but self-limited colitis. In epithelial cells, we observed the induction of small intestine-specific genes of the cryptdin family and colon-specific expression of serum amyloid A1 gene. This novel approach allows the identification of known and novel gene products involved in mucosal defense against luminal microorganisms and the associated inflammatory response.     

Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response

In the Cf-9/Avr9 gene-for-gene interaction, the Cf-9 resistance gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum, which expresses the corresponding pathogen-derived avirulence product Avr9. To understand R gene function and dissect the signaling mechanisms involved in the induction of plant defenses, we studied Cf-9/Avr9-dependent activation of protein kinases in transgenic Cf9 tobacco cell cultures. Using a modified in-gel kinase assay with histone as substrate, we identified a membrane-bound, calcium-dependent protein kinase (CDPK) that showed a shift in electrophoretic mobility from 68 to 70 kD within 5 min after Avr9 elicitor was added. This transition from the nonelicited to the elicited CDPK form was caused by a phosphorylation event and was verified when antibodies to CDPK were used for protein gel blot analysis. In addition, the interconversion of the corresponding CDPK forms could be induced in vitro in both directions by treatment with either phosphatase or ATP. In vitro protein kinase activity toward syntide-2 or histone with membrane extracts or gel-purified enzyme was dependent on Ca(2)+ content and was compromised by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) but not by its inactive isoform N-(6-aminohexyl)-1-naphthalenesulfonamide. In these assays, the CDPK activity in elicited samples, reflecting predominantly the phosphorylated 70-kD CDPK form, was greater than in nonelicited samples. Thus, Avr9/Cf-9-dependent phosphorylation and subsequent transition from the nonelicited to the elicited form correlate with the activation of a CDPK isoform after in vivo stimulation. Because that transition was not inhibited by W-7, the in vivo CDPK activation probably is not the result of autophosphorylation. Studies with pharmacological inhibitors indicated that the identified CDPK is independent of or is located upstream from a signaling pathway that is required for the Avr9-induced active oxygen species.     

Comprehensive analysis of protein-protein interactions between Arabidopsis MAPKs and MAPK kinases helps define potential MAPK signalling modules

The Arabidopsis genome encodes a 20-member gene family of mitogen-activated protein kinases (MPKs) but biological roles have only been identified for a small subset of these crucial signalling components. In particular, it is unclear how the MPKs may be organized into functional modules within the cell. To gain insight into their potential relationships, we used the yeast two-hybrid system to conduct a directed protein-protein interaction screen between all the Arabidopsis MPKs and their upstream activators (MAPK kinases; MKK). Novel interactions were also tested in vitro for enzyme-substrate functionality, using recombinant proteins. The resulting data confirm a number of earlier reported MKK-MPK relationships, but also reveal a more extensive pattern of interactions that should help to guide future analyses of MAPK signalling in plants.Key words: mitogen-activated protein kinase, mitogen-activated protein kinase kinase, yeast two-hybrid, phosphorylation, protein-protein interaction, ArabidopsisPlant genomes are notably rich in the number of protein kinase signalling components they encode^1–3 and it can therefore be anticipated that the associated signal transduction networks will be highly specialized and complex. Within the plant protein kinase super-family, the highly conserved Arabidopsis mitogen-activated protein kinases (MAPKs; MPKs) are represented by a 20-member family that is most closely related to the ERK class of metazoan MAPKs.⁴ This family includes three sub-families of MPKs whose activation domain carries a -TEY- motif, as well as a fourth, evolutionarily distinct -TDY- sub-family.^4,5 Dual-specificity MAPK kinases (MKK) serve as the canonical activators of MPKs through phosphorylation of both the threonine and tyrosine residues within the MPK activation loop -TXY- motif. The Arabidopsis genome encodes ten members of the MKK gene family, among which one (MKK10) lacks the fully conserved -S/T-X_3-5-S/T- motif that typifies eukaryotic MAPK kinases.⁵Numerous reports have provided evidence for the involvement of plant MPKs in a wide range of biotic and abiotic stress responses, as well as phytohormone signalling and developmental patterning, as recently reviewed in.⁶ However, defining functional MKK-MPK module combinations by connecting a particular activated MPK to a specific upstream MKK remains a challenge. Since there are precedents for activation of multiple MPKs by one MKK, as well as evidence for more than one MKK having the capability of activating a given MPK, there are many possible ways in which MKK-MPK signalling modules might potentially be configured. Phenotype-based forward genetic screens in Arabidopsis have provided relatively little insight into these relationships, with only one MPK (MPK4) being recovered as a loss-of-function mutant.⁷ The failure to recover mutations in the other MPK loci, or in any of the MKKs, in such screens could indicate that there is considerable functional redundancy within the MAPK signalling network, that the phenotypic consequences of a loss-of-function mutation are subtle or conditional, or that loss-of-function genotypes are non-viable.Since the nature of protein kinase/phosphatase activities depends on direct physical encounters between the enzyme and its target protein, we hypothesized that the ability of particular proteins to interact effectively with each other would define one level of specificity within the global Arabidopsis MKK/MPK network. To test this idea, we conducted a comprehensive directed yeast two-hybrid screen using the ten Arabidopsis MKKs as individual bait proteins, and each of the twenty MPKs as prey proteins. Several of the protein-protein interactions detected in this Y2H screen were also tested in direct phosphorylation assays in vitro, using recombinant proteins.Nine of the ten Arabidopsis MKK proteins were found to interact with at least one MPK protein in our Y2H assays (https://www.genevestigator.ethz.ch/gv/index.jsp). Its putative orthologue in Populus trichocarpa is similarly silent,^5,8 consistent with a gene that may be losing its biological functionality. Most other MKKs were found to interact with two or more MPK targets, and in several cases these results confirmed earlier reports of MKK-MPK interactions. For example, we found that MKK1 and MKK2, two closely related MAPKKs, both interacted with MPK 4 and with MPK11, a pair of paralogous MPKs. Interaction between MKK1 (MEK1) and MPK4 had already been observed in one of the first studies of plant MKK-MPK relationships,⁹ while a later study also found that MKK2 could interact with MPK4, among twelve MPKs surveyed.¹⁰ However, neither of these reports had examined MPK11. We could confirm by in vitro phosphorylation assays using “constitutively active” (CA) forms of recombinant MKK1 and MKK2 that both of these MKKs can phosphorylate recombinant MPK4, but, in contrast to the Y2H interaction pattern, both CAMKKs showed only very weak activity with MPK11 as a substrate (Fig. 2A and B).Open in a separate windowFigure 2Effects of incubation with recombinant GST-CAMKK proteins on protein phosphorylation of recombinant GST-MPKs. The constitutively active mutant forms of the MKKs were generated by QuickChange site-directed mutagenesis (Stratagene) and confirmed by sequencing. The conserved Ser or Thr residues in the activation loop in MKKs were replaced with acidic residues to create a “constitutively active” kinase (T218E and S224D for CAMKK1, T220D and T226E for CAMKK2, S221D and T227E for CAMKK6, S193E and S199D for CAMKK7, and S195E and S201E for CAMKK9). PCR amplicons of the full-length cDNAs corresponding to MPK2 (At1g59580), MPK4 (At4g01370), MPK6 (At2g43790), MPK10 (At3g59790), MPK11 (At1g01560), MPK13 (At1g07880), MPK17 (At2g01450), MPK20 (At2g42880), MKK1 (At4g26070), MKK2 (At4g29810), MKK6 (At5g56580), MKK7 (At1g18350) and MKK9 (At1g73500) were purified, digested with the appropriate restriction enzymes and subcloned in either the pGEX 4T-2 or pDEST15 vector, which expresses the recombinant protein with a N-terminal GST tag. Each of wild-type MAPK and mutant recombinant CAMKK1, CAMKK2, CAMKK6, CAMKK7 and CAMKK9 were expressed as glutathione S-transferase (GST) fusion proteins as previously described.¹⁹ For the in vitro phosphorylation assays, each GST-MPK (1 µg) was incubated in 25 µL of kinase reaction buffer (50 mM Tris-HCl, pH 7.5, 5 mM β-glycerolphosphate, 2 mM DTT, 10 mM MgCl₂, 0.1 mM Na₃VO₄, 0.1 mM ATP and 3 µCi of [γ-³²P] ATP) either with or without constitutively active GST-MKKs (0.3 µg) at 30°C for 30 min. The reaction was terminated by addition of concentrated SDS-PAGE sample buffer followed by boiling for 5 min. Reaction products were analyzed using SDS-PAGE, autoradiography, and CBB staining. (A) Phosphorylation of MPKs by incubation with CAMKK1. (B) Phosphorylation of MPKs by incubation with CAMKK2. (C) Phosphorylation of MPKs by incubation with CAMKK6. (D) Phosphorylation of MPK2 by incubation with CAMKK7. (E) Phosphorylation of MPKs by incubation with CAMKK9.

Table 1

Full-length cDNA clones corresponding to the open reading frame of each of the ten Arabidopsis MKK and twenty distinct MAPKs were isolated from Arabidopsis cDNA and cloned into a Gateway™ entry vector, either pENTR (Invitrogen) or pCR8 (Invitrogen)

Identification of Streptococcus thermophilus CNRZ368 genes involved in defense against superoxide stress

To better understand the defense mechanism of Streptococcus thermophilus against superoxide stress, molecular analysis of 10 menadione-sensitive mutants, obtained by insertional mutagenesis, was undertaken. This analysis allowed the identification of 10 genes that, with respect to their putative functions, were classified into five categories: (i) those involved in cell wall metabolism, (ii) those involved in exopolysaccharide translocation, (iii) those involved in RNA modification, (iv) those involved in iron homeostasis, and (v) those whose functions are still unknown. The behavior of the 10 menadione-sensitive mutants exposed to heat shock was investigated. Data from these experiments allowed us to distinguish genes whose action might be specific to oxidative stress defense (tgt, ossF, and ossG) from those whose action may be generalized to other stressful conditions (mreD, rodA, pbp2b, cpsX, and iscU). Among the mutants, two harbored an independently inserted copy of pGh9:ISS1 in two loci close to each other. More precisely, these two loci are homologous to the sufD and iscU genes, which are involved in the biosynthesis of iron-sulfur clusters. This region, called the suf region, was further characterized in S. thermophilus CNRZ368 by sequencing and by construction of DeltasufD and iscU(97) nonpolar mutants. The streptonigrin sensitivity levels of both mutants suggest that these two genes are involved in iron metabolism.     

Mechanisms involved in the bidirectional effects of protein kinase C activators on neutrophil responses to leukotriene B4

Three protein kinase C (PKC) activators (PMA, mezerein, and a diacylglycerol) had bidirectional effects on human polymorphonuclear neutrophil (PMN) degranulation responses to leukotriene (LT) B4. Lower concentrations of the three agents enhanced, whereas higher concentrations inhibited, release of lysozyme and beta-glucuronidase stimulated by the arachidonic acid metabolite. Contrastingly, the activators inhibited but never enhanced LTB4-induced Ca2+ transients. We examined the causes for these varying effects. Each PKC activator reduced PMN specific binding of [3H]LTB4. Scatchard analyses revealed that PMA (greater than or equal to 0.16 nM) decreased the number of high affinity LTB4 receptors. The receptor losses correlated closely with inhibition of Ca2+ transients. PMN pretreated with 0.5 nM PMA for 5 min retained approximately 50% of their high affinity LTB4 receptors. These cells responded to 10 nM LTB4 with reduced but still substantial rises in cytosolic Ca2+, enhanced PKC mobilization, and increased granule enzyme release. The latter two effects appeared calcium-dependent because sequential exposure to PMA and LTB4 did not synergistically stimulate PKC mobilization or degranulation in PMN that were: 1) Ca2(+)-depleted; 2) challenged with 5 nM PMA; or 3) treated with LTB4 for 5 min before PMA. Each of the latter treatments completely interfered with the extent or timing of LTB4-induced Ca2+ transients. Accordingly, we suggest that the response-specific, bidirectional effects of PKC activators on LTB4 result from two opposing mechanisms. First, PKC activators down-regulate LTB4 high affinity receptors and thereby reduce those PMN responses that are not elicited by activated PKC (i.e., Ca2+ transients). Second, LTB4, by elevating cytosolic Ca2+, increases the amount of PKC mobilized by PKC activators and thereby promotes PKC-dependent responses (e.g., degranulation). The two mechanisms may be pertinent to the bidirectional effects of PKC activators on various other agonists. Furthermore, PKC, by down-regulating receptors, may serve as a physiologic stop signal for terminating function and producing a poststimulatory state of desensitization.     

Decreased sulfation of cellular chondroitin sulfate in response to activators of protein kinase C

The sulfation of cellular chondroitin sulfate in human promyelocytic leukemia HL60 cells was inhibited by a number of phorbol diesters, which concurrently induced differentiation into monocytic cells. Inhibition was dependent on concentration, and was 90% complete at 10 nM 12-0-tetradecanoylphorbol-13-acetate (TPA), the most active ester. Maximal effects were seen within 2-4 hours following initiation of treatment. The degree of inhibition observed correlated well with the ability of the esters to induce differentiation, and with their reported affinity for a "receptor", identified as protein kinase C associated with certain lipids. Chondroitin sulfation was also inhibited in cells treated with sn-1,2-dioctanoylglycerol, a lipid which is considered to be an endogenous activator of protein kinase C. Our findings therefore indicate that monocytic differentiation of HL60 cells occurs subsequent to reduced glycosaminoglycan sulfation via activation of the calcium-activated, phospholipid-dependent protein kinase.     

Salicylic acid is part of the Mi-1-mediated defense response to root-knot nematode in tomato

The Mi-1 gene of tomato confers resistance against three species of root-knot nematode in tomato (Lycopersicon esculentum). Transformation of tomato carrying Mi-1 with a construct expressing NahG, which encodes salicylate hydroxylase, a bacterial enzyme that degrades salicylic acid (SA) to catechol, results in partial loss of resistance to root-knot nematodes. Exogenous SA was toxic to roots expressing NahG but not to control roots. This toxicity is most likely due to the production of catechol from SA, and we report here that 100 microM catechol is toxic to tomato roots. Benzothiadiazole, a SA analog, completely restores nematode resistance in Mi-1 roots transformed with NahG but does not confer resistance to susceptible tomato roots. The localized cell death produced by transient expression in Nicotiana benthamiana of Mi-DS4, a constitutively lethal chimera of Mi-1 with one of its homologs, was prevented by coexpression of NahG. These results indicate that SA is an important component of the signaling that leads to nematode resistance and the associated hypersensitive response.     

A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plants

The soil bacterium Bacillus subtilis is widely used in agriculture as a biocontrol agent able to protect plants from a variety of pathogens. Protection is thought to involve the formation of bacterial communities - biofilms - on the roots of the plants. Here we used confocal microscopy to visualize biofilms on the surface of the roots of tomato seedlings and demonstrated that biofilm formation requires genes governing the production of the extracellular matrix that holds cells together. We further show that biofilm formation was dependent on the sensor histidine kinase KinD and in particular on an extracellular CACHE domain implicated in small molecule sensing. Finally, we report that exudates of tomato roots strongly stimulated biofilm formation ex planta and that an abundant small molecule in the exudates, (L) -malic acid, was able to stimulate biofilm formation at high concentrations in a manner that depended on the KinD CACHE domain. We propose that small signalling molecules released by the roots of tomato plants are directly or indirectly recognized by KinD, triggering biofilm formation.