Effect of Thickening Agents on the In Vitro Antibacterial Activity of Vancomycin Hydrochloride Powder.

When vancomycin hydrochloride (VCM) powder mixes with xanthan gum-based thickening agents in food, lumps or other property-related changes may occur. Previous studies have reported delayed disintegration and elution of the drug and its adsorption on to xanthan gum, which is the main ingredient of thickened food products. If the addition of thickening agents can affect the antimicrobial activity of VCM powder as previously reported, it might interfere with the treatment of Clostridioides difficile infection (CDI). In this study, we investigated the effect of the addition of xanthan gum-based thickening agents on the antibacterial activity of VCM against Clostridioides difficile in vitro. The VCM concentration at 0 min after adding 3% Tsururinko Quickly (Clinico, Tokyo) to VCM powders (Shionogi, Osaka and Meiji Seika Pharma, Tokyo) was lower than that of the control [Shionogi: 65.15±35.57%, Meiji Seika Pharma: 77.00±15.81% (mean±standard deviation), ** p<0.01, Dunnet's test]. However, the VCM concentration at 30 min after the addition recovered to the control level. The drug susceptibility tests for C. difficile and Staphylococcus aureus using the disk diffusion method showed no effect of addition of 3% Tsururinko Quickly. Our in vitro evaluations showed that the addition of xanthan gum-based thickeners to VCM powders had a negligible effect on the treatment of CDI.

Cytoplasmic Kinase Network Mediates Defense Response to Spodoptera litura in Arabidopsis.

Plants defend against folivores by responding to folivore-derived elicitors following activation of signaling cascade networks. In Arabidopsis, HAK1, a receptor-like kinase, responds to polysaccharide elicitors (Frα) that are present in oral secretions of Spodoptera litura larvae to upregulate defense genes (e.g., PDF1.2) mediated through downstream cytoplasmic kinase PBL27. Here, we explored whether other protein kinases, including CPKs and CRKs, function with PBL27 in the intracellular signaling network for anti-herbivore responses. We showed that CRK2 and CRK3 were found to interact with PBL27, but CPKs did not. Although transcripts of PDF1.2 were upregulated in leaves of wild-type Arabidopsis plants in response to mechanical damage with Frα, this failed in CRK2- and PBL27-deficient mutant plants, indicating that the CRK2/PBL27 system is predominantly responsible for the Frα-responsive transcription of PDF1.2 in S. litura-damaged plants. In addition to CRK2-phosphorylated ERF13, as shown previously, ethylene signaling in connection to CRK2-phosphorylated PBL27 was predicted to be responsible for transcriptional regulation of a gene for ethylene response factor 13 (ERF13). Taken together, these findings show that CRK2 regulates not only ERF13 phosphorylation but also PBL27-dependent de novo synthesis of ERF13, thus determining active defense traits against S. litura larvae via transcriptional regulation of PDF1.2.

Identification of a new gibberellin receptor agonist, diphegaractin, by a cell-free chemical screening system.

Gibberellin (GA) is a phytohormone that regulates various developmental processes during the plant life cycle. In this study, we identify a new GA agonist, diphegaractin, using a wheat cell-free based drug screening system with grape GA receptor. A GA-dependent interaction assay system using GA receptors and DELLA proteins from Vitis vinifera was constructed using AlphaScreen technology and cell-free produced proteins. From the chemical compound library, diphegaractin was found to enhance the interactions between GA receptors and DELLA proteins from grape in vitro. In grapes, we found that diphegaractin induces elongation of the bunch and increases the sugar concentration of grape berries. Furthermore, diphegaractin shows GA-like activity, including promotion of root elongation in lettuce and Arabidopsis, as well as reducing peel pigmentation and suppressing peel puffing in citrus fruit. To the best of our knowledge, this study is the first to successfully identify a GA receptor agonist showing GA-like activity in agricultural plants using an in vitro molecular-targeted drug screening system.

JUL1, Ring-Type E3 Ubiquitin Ligase, Is Involved in Transcriptional Reprogramming for ERF15-Mediated Gene Regulation.

JAV1-associated ubiquitin ligase 1 (JUL1) is a RING-type E3 ubiquitin ligase that catalyzes ubiquitination of JAV1, a jasmonate signaling repressor, in Arabidopsis thaliana in response to herbivore attack. Here we present a new insight into the nature of JUL1 as a multi-targeting enzyme for not only JAV1 but also transcription factors (TFs) screened using in vitro and in vivo protein interaction assays. Reporter assays using protoplasts showed that the JUL1-interacting TFs (JiTFs), including ERF15, bZIP53 and ORA59, were involved in transcriptional activation of jasmonate-responsive PDF1.2 and abscisic acid-responsive GEA6. Likewise, assays using mutant plants suggested that the 3 JiTFs were indeed responsible for transcriptional regulation of PDF1.2 and/or GEA6, and ERF15 and ORA59 were substantially responsible for the anti-herbivore trait. In vitro protein ubiqutination assays showed that JUL1 catalyzed ubiqutination of JAV1 but not any of the TFs. This was in accord with the finding that JUL1 abolished JAV1's interference with ERF15 function, according to the reporter assay. Moreover, of great interest is our finding that ERF15 but not bZIP53 or ORA59 serves as a scaffold for the JAV1/JUL1 system, indicating that there is narrow selectivity of the transcriptional reprogramming by the JAV1/JUL1 system.

Immune gene activation by NPR and TGA transcriptional regulators in the model monocot Brachypodium distachyon.

The nonexpressor of pathogenesis-related (NPR) gene family is well known to play a crucial role in transactivation of TGA transcription factors for salicylic acid (SA)-responsive genes, including pathogenesis-related protein 1 (PR1), during plants' immune response after pathogen attack in the model dicot Arabidopsis thaliana. However, little is known about NPR gene functions in monocots. We therefore explored the functions of NPRs in SA signaling in the model monocot Brachypodium distachyon. BdNPR1 and BdNPR2/3 share structural similarities with A. thaliana AtNPR1/2 and AtNPR3/4 subfamilies, respectively. The transcript level of BdNPR2 but not BdNPR1/3 appeared to be positively regulated in leaves in response to methyl salicylate. Reporter assays in protoplasts showed that BdNPR2 positively regulated BdTGA1-mediated activation of PR1. This transactivation occurred in an SA-dependent manner through SA binding at Arg468 of BdNPR2. In contrast, BdNPR1 functioned as a suppressor of BdNPR2/BdTGA1-mediated transcription of PR1. Collectively, our findings reveal that the TGA-promoted transcription of SA-inducible PR1 is orchestrated by the activator BdNPR2 and the repressor BdNPR1, which function competitively in B. distachyon.

A simple method for labeling proteins and antibodies with biotin using the proximity biotinylation enzyme TurboID.

Proteins and antibodies labeled with biotin have been widely used for protein analysis, enzyme immunoassays, and diagnoses. Presently, they are prepared using either a chemical reaction involving a biotin N-hydroxysuccinimide (NHS) ester compound or by enzymatic biotin ligation using a combination of a biotinylation-peptide tag and Escherichia coli BirA. However, these methods are relatively complicated. Recently BirA was improved to TurboID, a highly active enzyme for proximity labeling with biotin. Here, we demonstrate a novel simple biotin labeling method for proteins and antibodies using TurboID. Purified TurboID was mixed with a protein or an antibody in the presence of biotin and ATP in the general biochemical buffer condition, followed by biotin labeling. Biotin labeling sites by TurboID were found on the surface of green fluorescent protein. Biotin labeling of IκBα by TurboID indicated its binding to RelA. Furthermore, TurboID-dependent biotin labeling of monoclonal antibodies from rabbits and mice could be directly used for immunoblotting detection of specific proteins without the purification step. These results indicate that TurboID provides a very useful and simple method for biotin labeling of functional proteins.

The rice wound-inducible transcription factor RERJ1 sharing same signal transduction pathway with OsMYC2 is necessary for defense response to herbivory and bacterial blight.

KEY MESSAGE: This study describes biological functions of the bHLH transcription factor RERJ1 involved in the jasmonate response and the related defense-associated metabolic pathways in rice, with particular focus on deciphering the regulatory mechanisms underlying stress-induced volatile emission and herbivory resistance. RERJ1 is rapidly and drastically induced by wounding and jasmonate treatment but its biological function remains unknown as yet. Here we provide evidence of the biological function of RERJ1 in plant defense, specifically in response to herbivory and pathogen attack, and offer insights into the RERJ1-mediated regulation of metabolic pathways of specialized defense compounds, such as monoterpene linalool, in possible collaboration with OsMYC2-a well-known master regulator in jasmonate signaling. In rice (Oryza sativa L.), the basic helix-loop-helix (bHLH) family transcription factor RERJ1 is induced under environmental stresses, such as wounding and drought, which are closely linked to jasmonate (JA) accumulation. Here, we investigated the biological function of RERJ1 in response to biotic stresses, such as herbivory and pathogen infection, using an RERJ1-defective mutant. Transcriptome analysis of the rerj1-Tos17 mutant revealed that RERJ1 regulated the expression of a typical family of conserved JA-responsive genes (e.g., terpene synthases, proteinase inhibitors, and jasmonate ZIM domain proteins). Upon exposure to armyworm attack, the rerj1-Tos17 mutant exhibited more severe damage than the wildtype, and significant weight gain of the larvae fed on the mutant was observed. Upon Xanthomonas oryzae infection, the rerj1-Tos17 mutant developed more severe symptoms than the wildtype. Among RERJ1-regulated terpene synthases, linalool synthase expression was markedly disrupted and linalool emission after wounding was significantly decreased in the rerj1-Tos17 mutant. RERJ1 appears to interact with OsMYC2-a master regulator of JA signaling-and many OsJAZ proteins, although no obvious epistatic interaction was detected between them at the transcriptional level. These results indicate that RERJ1 is involved in the transcriptional induction of JA-mediated stress-responsive genes via physical association with OsMYC2 and mediates defense against herbivory and bacterial infection through JA signaling.

Protein-protein interactions between jasmonate-related master regulator MYC and transcriptional mediator MED25 depend on a short binding domain.

A network of protein-protein interactions (PPI) is involved in the activation of (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), a plant hormone that regulates plant defense responses as well as plant growth and development. In the absence of JA-Ile, inhibitory protein jasmonate-ZIM-domain (JAZ) represses JA-related transcription factors, including a master regulator, MYC. In contrast, when JA-Ile accumulates in response to environmental stresses, PPI occurs between JAZ and the F-box protein COI1, which triggers JAZ degradation, resulting in derepressed MYC that can interact with the transcriptional mediator MED25 and upregulate JA-Ile-related gene expression. Activated JA signaling is eventually suppressed through the catabolism of JA-Ile and feedback suppression by JAZ splice variants containing a cryptic MYC-interacting domain (CMID). However, the detailed structural basis of some PPIs involved in JA-Ile signaling remains unclear. Herein, we analyzed PPI between MYC3 and MED25, focusing on the key interactions that activate the JA-Ile signaling pathway. Biochemical assays revealed that a short binding domain of MED25 (CMIDM) is responsible for the interaction with MYC, and that a bipartite interaction is critical for the formation of a stable complex. We also show the mode of interaction between MED25 and MYC is closely related to that of CMID and MYC. In addition, quantitative analyses on the binding of MYC3-JAZs and MYC3-MED25 revealed the order of binding affinity as JAZJas < MED25CMIDM < JAZCMID, suggesting a mechanism for how the transcriptional machinery causes activation and negative feedback regulation during jasmonate signaling. These results further illuminate the transcriptional machinery responsible for JA-Ile signaling.

Deciphering OPDA Signaling Components in the Momilactone-Producing Moss Calohypnum plumiforme.

Jasmonic acid (JA) and its biologically active form jasmonoyl-L-isoleucine (JA-Ile) regulate defense responses to various environmental stresses and developmental processes in plants. JA and JA-Ile are synthesized from α-linolenic acids derived from membrane lipids via 12-oxo-phytodienoic acid (OPDA). In the presence of JA-Ile, the COI1 receptor physically interacts with JAZ repressors, leading to their degradation, resulting in the transcription of JA-responsive genes by MYC transcription factors. Although the biosynthesis of JA-Ile is conserved in vascular plants, it is not recognized by COI1 in bryophytes and is not biologically active. In the liverwort Marchantia polymorpha, dinor-OPDA (dn-OPDA), a homolog of OPDA with two fewer carbons, and its isomer dn-iso-OPDA accumulate after wounding and are recognized by COI1 to activate downstream signaling. The moss Calohypnum plumiforme produces the antimicrobial-specialized metabolites, momilactones. It has been reported that JA and JA-Ile are not detected in C. plumiforme and that OPDA, but not JA, can induce momilactone accumulation and the expression of these biosynthetic genes, suggesting that OPDA or its derivative is a biologically active molecule in C. plumiforme that induces chemical defense. In the present study, we investigated the biological functions of OPDA and its derivatives in C. plumiforme. Searching for the components potentially involving oxylipin signaling from transcriptomic and genomic data revealed that two COI1, three JAZ, and two MYC genes were present. Quantification analyses revealed that OPDA and its isomer iso-OPDA accumulated in larger amounts than dn-OPDA and dn-iso-OPDA after wounding. Moreover, exogenously applied OPDA, dn-OPDA, or dn-iso-OPDA induced the transcription of JAZ genes. These results imply that OPDA, dn-OPDA, and/or their isomers potentially act as biologically active molecules to induce the signaling downstream of COI1-JAZ. Furthermore, co-immunoprecipitation analysis showed the physical interaction between JAZs and MYCs, indicating the functional conservation of JAZs in C. plumiforme with other plants. These results suggest that COI1-JAZ-MYC mediated signaling is conserved and functional in C. plumiforme.

Expression of LhFT1, the Flowering Inducer of Asiatic Hybrid Lily, in the Bulb Scales.

Asiatic hybrid lily leaves emerge from their bulbs in spring, after cold exposure in winter, and the plant then blooms in early summer. We identified four FLOWERING LOCUS T (FT)-like genes, LhFT1, LhFT4, LhFT6, and LhFT8, from an Asiatic hybrid lily. Floral bud differentiation initiated within bulbs before the emergence of leaves. LhFT genes were mainly expressed in bulb scales, and hardly in leaves, in which the FT-like genes of many plants are expressed in response to environmental signals. LhFT1 was expressed in bulb scales after vernalization and was correlated to flower bud initiation in two cultivars with different flowering behaviors. LhFT8 was upregulated in bulb scales after cold exposure and three alternative splicing variants with a nonsense codon were simultaneously expressed. LhFT6 was upregulated in bulb scales after flower initiation, whereas LhFT4 was expressed constantly in all organs. LhFT1 overexpression complemented the late-flowering phenotype of Arabidopsis ft-10, whereas that of LhFT8 did so partly. LhFT4 and LhFT6 overexpression could not complement. Yeast two-hybrid and in vitro analyses showed that the LhFT1 protein interacted with the LhFD protein. LhFT6 and LhFT8 proteins also interacted with LhFD, as observed in AlphaScreen assay. Based on these results, we revealed that LhFT1 acts as a floral activator during floral bud initiation in Asiatic hybrid lilies. However, the biological functions of LhFT4, LhFT6, and LhFT8 remain unclear.

Characterization of mitochondrial carrier proteins of malaria parasite Plasmodium falciparum based on in vitro translation and reconstitution.

Members of the mitochondrial carrier (MC) family of membrane transporters play important roles in cellular metabolism. We previously established an in vitro reconstitution system for membrane transporters based on wheat germ cell-free translation system. We have now applied this reconstitution system to the comparative analysis of MC proteins from the malaria parasite Plasmodium falciparum and Saccharomyces cerevisiae. We synthesized twelve putative P. falciparum MCs and determined the transport activities of four of these proteins including PF3D7_1037300 protein (ADP/ATP translocator), PF3D7_1004800 protein (ADP/ATP translocator), PF3D7_1202200 protein (phosphate carrier), and PF3D7_1241600 protein (S-adenosylmethionine transporter). In addition, we tested the effect of cardiolipin on the activity of MC proteins. The transport activities of the yeast MCs, ScAac2p, ScGgc1p, ScDic1p, ScPic1p, and ScSam5p, which localize to the mitochondrial inner membrane, were increased by cardiolipin supplementation, whereas that of ScAnt1p, which localizes to the peroxisome membrane, was not significantly affected. Together, this indicates that the functional properties of the reconstituted MCs reflect the lipid content of their native membranes. Except for PF3D7_1241600 protein, these P. falciparum proteins manifested cardiolipin-dependent transport activities. Immunofluorescence analysis showed that PF3D7_1241600 protein is not mainly localized to the mitochondria of P. falciparum cells. We thus revealed the functions of four MC proteins of the malaria parasite and the effects of cardiolipin on their activities.

Soy and Arabidopsis receptor-like kinases respond to polysaccharide signals from Spodoptera species and mediate herbivore resistance.

Plants respond to herbivory by perceiving herbivore danger signal(s) (HDS(s)), including "elicitors", that are present in herbivores' oral secretions (OS) and act to induce defense responses. However, little is known about HDS-specific molecules and intracellular signaling. Here we explored soybean receptor-like kinases (RLKs) as candidates that might mediate HDS-associated RLKs' (HAKs') actions in leaves in response to OS extracted from larvae of a generalist herbivore, Spodoptera litura. Fractionation of OS yielded Frα, which consisted of polysaccharides. The GmHAKs composed of their respective homomultimers scarcely interacted with Frα. Moreover, Arabidopsis HAK1 homomultimers interacted with cytoplasmic signaling molecule PBL27, resulting in herbivory resistance, in an ethylene-dependent manner. Altogether, our findings suggest that HAKs are herbivore-specific RLKs mediating HDS-transmitting, intracellular signaling through interaction with PBL27 and the subsequent ethylene signaling for plant defense responses in host plants.

AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions.

Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4CRBN complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein-protein interactions.

Proteins in a cell need to interact with each other to perform the many tasks required for organisms to thrive. A technique called proximity biotinylation helps scientists to pinpoint the identity of the proteins that partner together. It relies on attaching an enzyme (either BioID or TurboID) to a protein of interest; when a partner protein comes in close contact with this construct, the enzyme can attach a chemical tag called biotin to it. The tagged proteins can then be identified, revealing which molecules interact with the protein of interest. Although BioID and TurboID are useful tools, they have some limitations. Experiments using BioID take more than 16 hours to complete and require high levels of biotin to be added to the cells. TurboID is more active than BioID and is able to label proteins within ten minutes. However, under certain conditions, it is also more likely to be toxic for the cell, or to make mistakes and tag proteins that do not interact with the protein of interest. To address these issues, Kido et al. developed AirID, a new enzyme for proximity biotinylation. Experiments were then conducted to test how well AirID would perform, using proteins of interest whose partners were already known. These confirm that AirID was able to label partner proteins in human cells; compared with TurboID, it was also less likely to mistakenly tag non-partners or to kill the cells, even over long periods. The results by Kido et al. demonstrate that AirID is suitable for proximity biotinylation experiments in cells. Unlike BioID and TurboID, the enzyme may also have the potential to be used for long-lasting experiments in living organisms, since it is less toxic for cells over time.

Production of a rabbit monoclonal antibody for highly sensitive detection of citrus mosaic virus and related viruses.

Citrus mosaic virus (CiMV) is one of the causal viruses of citrus mosaic disease in satsuma mandarins (Citrus unshiu). Prompt detection of trees infected with citrus mosaic disease is important for preventing the spread of this disease. Although rabbit monoclonal antibodies (mAbs) exhibit high specificity and affinity, their applicability is limited by technical difficulties associated with the hybridoma-based technology used for raising these mAbs. Here, we demonstrate a feasible CiMV detection system using a specific rabbit mAb against CiMV coat protein. A conserved peptide fragment of the small subunit of CiMV coat protein was designed and used to immunize rabbits. Antigen-specific antibody-producing cells were identified by the immunospot array assay on a chip method. After cloning of variable regions in heavy or light chain by RT-PCR from these cells, a gene set of 33 mAbs was constructed and these mAbs were produced using Expi293F cells. Screening with the AlphaScreen system revealed eight mAbs exhibiting strong interaction with the antigen peptide. From subsequent sequence analysis, they were grouped into three mAbs denoted as No. 4, 9, and 20. Surface plasmon resonance analysis demonstrated that the affinity of these mAbs for the antigen peptide ranged from 8.7 × 10-10 to 5.5 × 10-11 M. In addition to CiMV, mAb No. 9 and 20 could detect CiMV-related viruses in leaf extracts by ELISA. Further, mAb No. 20 showed a high sensitivity to CiMV and CiMV-related viruses, simply by dot blot analysis. The anti-CiMV rabbit mAbs obtained in this study are envisioned to be extremely useful for practical applications of CiMV detection, such as in a virus detection kit.

Substrate specificity of plastid phosphate transporters in a non-photosynthetic diatom and its implication in evolution of red alga-derived complex plastids.

The triose phosphate transporter (TPT) is one of the prerequisites to exchange metabolites between the cytosol and plastids. In this study, we demonstrated that the four plastid TPT homologues in the non-photosynthetic diatom Nitzschia sp. NIES-3581 were highly likely integrated into plastid envelope membranes similar to counterparts in the model photosynthetic diatom Phaeodactylum tricornutum, in terms of target membranes and C-terminal orientations. Three of the four Nitzschia TPT homologues are capable of transporting various metabolites into proteo-liposomes including triose phosphates (TPs) and phosphoenolpyruvate (PEP), the transport substrates sufficient to support the metabolic pathways retained in the non-photosynthetic diatom plastid. Phylogenetic analysis of TPTs and closely related transporter proteins indicated that diatoms and other algae with red alga-derived complex plastids possess only TPT homologues but lack homologues of the glucose 6-phosphate transporter (GPT), xylulose 5-phosphate transporter (XPT), and phosphoenolpyruvate transporter (PPT). Comparative sequence analysis suggests that many TPT homologues of red alga-derived complex plastids potentially have the ability to transport mainly TPs and PEP. TPTs transporting both TPs and PEP highly likely mediate a metabolic crosstalk between a red alga-derived complex plastid and the cytosol in photosynthetic and non-photosynthetic species, which explains the lack of PPTs in all the lineages with red alga-derived complex plastids. The PEP-transporting TPTs might have emerged in an early phase of endosymbiosis between a red alga and a eukaryote host, given the broad distribution of that type of transporters in all branches of red alga-derived complex plastid-bearing lineages, and have probably played a key role in the establishment and retention of a controllable, intracellular metabolic connection in those organisms.

Tyrosine Kinase-Dependent Defense Responses Against Herbivory in Arabidopsis.

Tyrosine (Tyr) phosphorylation (TP) is important for promotion of plants' signaling. Arabidopsis calcium-dependent protein kinase related protein kinases (CRK2 and CRK3) phosphorylate Tyr residues of a subset of transcription factors (TFs), including herbivory-responsive ethylene response factor 13 (ERF13), but the in vivo functions of these kinases in plant defense responses and development remain to be clarified. We show that when CRKs were coexpressed with ERF13 in Arabidopsis leaf protoplasts, the transcription activity regulated via ERF13 was elevated by CRK2 but not CRK3 or their kinase-dead form mutants. Moreover, this elevation was abolished when a Tyr-phosphorylation mutant of ERF was coexpressed with CRK2, indicating that CRK2 serves as an effector of ERF13 mediated by Tyr-phosphorylation. Moreover, CRK2 and CRK3 acted as effectors of RAP2.6 and WRKY14, respectively. CRK-overexpressing lines and knockout mutants of Arabidopsis plants showed increased and decreased expression levels of the defensin gene PDF1.2 in leaves, respectively, conferring on the plants modulated defense properties against the generalist herbivore Spodoptera litura. However, these lines did not show any obvious developmental defects, indicating that CRKs play a role in defense responses but not in the ordinary growth or development of plants. Transcription of both CRK2 and CRK3 was positively regulated by jasmonate signaling and abscisic acid (ABA) signaling upon herbivory. Our findings suggest that these phytohormone-responsive CRKs work coordinately for plant defense responses via Tyr phosphorylation of herbivory-responsive regulators.

Novel lineage-specific transmembrane ß-barrel proteins in the endoplasmic reticulum of Entamoeba histolytica.

ß-barrel outer membrane proteins (BOMPs) are essential components of outer membranes of Gram-negative bacteria and endosymbiotic organelles, usually involved in the transport of proteins and substrates across the membrane. Based on the analysis of our in silico BOMP predictor data for the Entamoeba histolytica genome, we detected a new transmembrane ß-barrel domain-containing protein, EHI_192610. Sequence analysis revealed that this protein is unique to Entamoeba species, and it exclusively clusters with a homolog, EHI_099780, which is similarly lineage specific. Both proteins possess an N-terminal signal peptide sequence as well as multiple repeats that contain dyad hydrophobic periodicities. Data from immunofluorescence assay of trophozoites expressing the respective candidates showed the absence of colocalization with mitosomal marker, and interestingly demonstrated partial colocalization with endoplasmic reticulum (ER) proteins instead. Integration to organellar membrane was supported by carbonate fractionation assay and immunoelectron microscopy. CD analysis of reconstituted proteoliposomes containing EHI_192610 showed a spectrum demonstrating a predominant ß-sheet structure, suggesting that this protein is ß-strand rich. Furthermore, the presence of repeat regions with predicted transmembrane ß-strand pairs in both EHI_192610 and EHI_099780, is consistent with the hypothesis that BOMPs originated from the amplification of ßß-hairpin modules, suggesting that the two Entamoeba-specific proteins are novel ß-barrels, intriguingly localized partially to the ER membrane.

The Ring-Type E3 Ubiquitin Ligase JUL1 Targets the VQ-Motif Protein JAV1 to Coordinate Jasmonate Signaling.

Jasmonates regulate plant defense and development. In Arabidopsis (Arabidopsis thaliana), JASMONATE-ASSOCIATED VQ-MOTIF GENE1 (JAV1/VQ22) is a repressor of jasmonate-mediated defense responses and is degraded through the ubiquitin-26S proteasome system after herbivory. We found that JAV1-ASSOCIATED UBIQUITIN LIGASE1 (JUL1), a RING-type E3 ubiquitin ligase, interacted with JAV1. JUL1 interacted with JAV1 in the nucleus to ubiquitinate JAV1, leading to proteasomal degradation of JAV1. The transcript levels of JUL1 and JAV1 were coordinately and positively regulated by the CORONATINE INSENSITIVE1-dependent signaling pathway in the jasmonate signaling network, but in a manner that was not dependent on CORONATINE INSENSITIVE1-mediated signaling upon herbivory by Spodoptera litura Gain or loss of function of JUL1 modulated the expression levels of the defensin gene PDF1.2 in leaves, conferring on the plants various defense properties against the generalist herbivore S. litura Because neither the JUL1 mutant nor overexpression lines showed any obvious developmental defects, we concluded that the JAV1/JUL1 system functions as a specific coordinator of reprogramming of plant defense responses. Altogether, our findings offer insight into the mechanisms by which the JAV1/JUL1 system acts specifically to coordinate plant defense responses without interfering with plant development or growth.

Identification of new abscisic acid receptor agonists using a wheat cell-free based drug screening system.

Abscisic acid (ABA) is the main phytohormone involved in abiotic stress response and its adaptation, and is a candidate agrichemical. Consequently, several agonists of ABA have been developed using the yeast two-hybrid system. Here, we describe a novel cell-free-based drug screening approach for the development and validation of ABA receptor agonists. Biochemical validation of this approach between 14 ABA receptors (PYR/PYL/RCARs) and 7 type 2C-A protein phosphatases (PP2CAs) revealed the same interactions as those of previous proteome data, except for nine new interactions. By chemical screening using this approach, we identified two novel ABA receptor agonists, JFA1 (julolidine and fluorine containing ABA receptor activator 1) and JFA2 as its analog. The results of biochemical validation for this approach and biological analysis suggested that JFA1 and JFA2 inhibit seed germination and cotyledon greening of seedlings by activating PYR1 and PYL1, and that JFA2 enhanced drought tolerance without inhibiting root growth by activating not only PYR1 and PYL1 but also PYL5. Thus, our approach was useful for the development of ABA receptor agonists and their validation.

Cell-Free Synthesis of Plant Receptor Kinases.

The wheat germ cell-free protein synthesis system has been used as a eukaryotic protein production system since it was first reported in 1964. Although initially the productivity of this system was not very high, it has now become one of the most versatile protein production systems, thanks to the enhancements made by several groups. In this chapter, we report a protein production method for plant receptor kinases using the wheat cell-free system. We describe a method for the preparation of a cell-free extract from wheat germ, the split-primer PCR method for preparation of transcription templates, and the bilayer cell-free protein synthesis method.