Contribution of salicylic acid glucosyltransferase, OsSGT1, to chemically induced disease resistance in rice plants

Systemic acquired resistance (SAR), a natural disease response in plants, can be induced chemically. Salicylic acid (SA) acts as a key endogenous signaling molecule that mediates SAR in dicotyledonous plants. However, the role of SA in monocotyledonous plants has yet to be elucidated. In this study, the mode of action of the agrochemical protectant chemical probenazole was assessed by microarray-based determination of gene expression. Cloning and characterization of the most highly activated probenazole-responsive gene revealed that it encodes UDP-glucose:SA glucosyltransferase (OsSGT1) , which catalyzes the conversion of free SA into SA O- β-glucoside (SAG). We found that SAG accumulated in rice leaf tissue following treatment with probenazole or 2,6-dichloroisonicotinic acid. A putative OsSGT1 gene from the rice cultivar Akitakomachi was cloned and the gene product expressed in Escherichia coli was characterized, and the results suggested that probenazole-responsive OsSGT1 is involved in the production of SAG. Furthermore, RNAi-mediated silencing of the OsSGT1 gene significantly reduced the probenazole-dependent development of resistance against blast disease, further supporting the suggestion that OsSGT1 is a key mediator of development of chemically induced disease resistance. The OsSGT1 gene may contribute to the SA signaling mechanism by inducing up-regulation of SAG in rice plants.     

Expression and stability of the nontoxic component of the botulinum toxin complex

Clostridium botulinum produces botulinum neurotoxin (BoNT) as a large toxin complex associated with nontoxic-nonhemagglutinin (NTNHA) and/or hemagglutinin components. In the present study, high-level expression of full-length (1197 amino acids) rNTNHA from C. botulinum serotype D strain 4947 (D-4947) was achieved in an Escherichia coli system. Spontaneous nicking of the rNTNHA at a specific site was observed during long-term incubation in the presence of protease inhibitors; this was also observed in natural NTNHA. The rNTNHA assembled with isolated D-4947 BoNT with molar ratio 1:1 to form a toxin complex. The reconstituted toxin complex exhibited dramatic resistance to proteolysis by pepsin or trypsin at high concentrations, despite the fact that the isolated BoNT and rNTNHA proteins were both easily degraded. We provide definitive evidence that NTNHA plays a crucial role in protecting BoNT, which is an oral toxin, from digestion by proteases common in the stomach and intestine.     

Mechanical stretch stimulates integrin αVβ3-mediated collagen expression in human anterior cruciate ligament cells

Biomechanical stimuli have fundamental roles in the maintenance and remodeling of ligaments including collagen gene expressions. Mechanical stretching signals are mainly transduced by cell adhesion molecules such as integrins. However, the relationships between stress-induced collagen expressions and integrin-mediated cellular behaviors are still unclear in anterior cruciate ligament cells. Here, we focused on the stretch-related responses of different cells derived from the ligament-to-bone interface and midsubstance regions of human anterior cruciate ligaments. Chondroblastic interface cells easily lost their potential to produce collagen genes in non-stretched conditions, rather than fibroblastic midsubstance cells. Uni-axial mechanical stretches increased the type I collagen gene expression of interface and midsubstance cells up to 14- and 6-fold levels of each non-stretched control, respectively. Mechanical stretches also activated the stress fiber formation by shifting the distribution of integrin αVβ3 to the peripheral edges in both interface and midsubstance cells. In addition, integrin αVβ3 colocalized with phosphorylated focal adhesion kinase in stretched cells. Functional blocking analyses using anti-integrin antibodies revealed that the stretch-activated collagen gene expressions on fibronectin were dependent on integrin αVβ3-mediated cellular adhesions in the interface and midsubstance cells. These findings suggest that the integrin αVβ3-mediated stretch signal transduction might have a key role to stimulate collagen gene expression in human anterior cruciate ligament, especially in the ligament-to-bone interface.     

A dual resistance gene system prevents infection by three distinct pathogens

Colletotrichum higginsianum causes typical anthracnose lesions on the leaves, petioles, and stems of cruciferous plants. Inoculation of Arabidopsis thaliana ecotype Columbia leaves with C. higginsianum results in fungal growth and disease symptoms reminiscent of those induced in other cruciferous plants. We performed map-based cloning and natural variation analysis of 19 A. thaliana ecotypes to identify a dominant resistance locus against C. higginsianum. We found that the A. thaliana RCH2 (for recognition of C. higginsianum) locus encodes two NB-LRR proteins, both of which are required for resistance to C. higginsianum in the A. thaliana ecotype Ws-0. Both proteins are well-characterized R proteins involved in resistance against bacterial pathogens; RRS1 (resistance to Ralstonia solanacearum 1) confers resistance to strain Rs1000 of R. solanacearum and RPS4 to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 (Pst-avrRps4). Furthermore, we found that both RRS1-Ws and RPS4-Ws genes are required for resistance to Pst-avrRps4 and to Rs1002 R. solanacearum. We therefore demonstrate that a pair of neighboring genes, RRS1-Ws and RPS4-Ws, function cooperatively as a dual R-gene system against at least three distinct pathogens.Key words: R gene, RPS4, RRS1, Colletotrichum higginsianum, Pseudomonas syringae, Ralstonia solanacearumPlants are exposed to various types of potentially invasive organisms, including viruses, bacteria, fungi, nematodes and protozoa, but are able to defend themselves by activating multiple defense mechanisms. The gene-for-gene hypothesis¹ provides a mechanism for specific recognition of the pathogen by the plant. This recognition is mediated by direct or indirect interactions between the product of a plant resistance (R) gene and the corresponding effectors encoded by avirulence genes in the pathogen.² Most R-genes encode non-membrane proteins that contain a conserved nucleotide-binding (NB) site and a carboxy-terminal leucine-rich repeat (LRR) domain.The A. thaliana genome contains about 150 genes coding for NB-LRR-containing proteins.³ This is far less than the number of genes that would be required to respond individually and specifically to all of its potential pathogens. However, plants may have been able to limit the number of required NB-LRR-encoding genes if host proteins perceive sets of distinct pathogens.⁴Colletotrichum species cause devastating anthracnose diseases in a large number of agronomically important crops. These diseases can often be controlled by introduction of genetic resistance traits, but the molecular components of resistance remain unknown. Inoculation of A. thaliana ecotype Columbia (Col-0) leaves with Colletotrichum higginsianum results in fungal growth and disease symptoms reminiscent of those induced in other cruciferous plants.^5,6 Inoculation of a large number of ecotypes with isolates of C. higginsianum showed that A. thaliana has at least two dominant resistance gene loci, designated RCH1 and RCH2 (for recognition of C. higginsianum), indicating that A. thaliana resistance to C. higginsianum is controlled by a “gene-for-gene” interaction.⁵ In a previous study, we identified a single putative R locus, RCH1 on the top of chromosome 4, in the C. higginsianum-resistant A. thaliana ecotype Eil-0.⁵In the present study, the locus named RCH2 maps in an extensive cluster of disease-resistance loci known as MRC-J in the A. thaliana ecotype Ws-0. By analyzing natural variations within the MRC-J region, we found that alleles of RRS1 (resistance to Ralstonia solanacearum 1) from susceptible ecotypes contain single nucleotide polymorphisms that may affect the encoded protein. Consistent with this finding, two susceptible mutants, rrs1-1 and rrs1–2, were identified by screening a T-DNA-tagged mutant library for the loss of resistance to C. higginsianum. The screening identified an additional susceptible mutant (rps4-21), which has a 5-bp deletion in the neighboring gene, RPS4-Ws, a well-characterized R gene that provides resistance to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 (Pst-avrRps4). To assess if RRS1-Ws and RPS4-Ws function in concert, we generated an rps4-21/rrs1-1 double mutant by crossing rps4-21 and rrs1-1 mutants. The susceptibility levels of rps4-21/rrs1-1 double mutant to C. higginsianum were similar to that exhibited by the single mutants, suggesting that RRS1-Ws and RPS-4-Ws function cooperatively. We also found that both RRS1 and RPS4 are required for resistance to R. solanacearum and Pst-avrRps4. Thus, these two adjacent R genes confer resistance, in tandem or individually, to three distinct pathogens with very different infection strategies and virulence mechanisms (Fig. 1).Open in a separate windowFigure 1RPS4 and RRS1 function as a dual resistance gene system that prevents infection by three distinct pathogens (Colletotrichum higginsianum, Ralstonia solanacearum and Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4).Several examples of two NB-LRR genes acting cooperatively to confer resistance against a pathogen have been reported. For example, A. thaliana RPP2A and RPP2B reside adjacently in the RPP2 locus.⁷ Blast resistance in Pikm-containing rice is conferred by a combination of two NB-LRR encoding genes, Pikm1-TS and Pikm2-TS.⁸ Pi5-mediated resistance against rice blast requires two NB-LRR-encoding genes.⁹ It is not known whether these NB-LRR genes function cooperatively or independently. Because of structural similarity with RRS1/RPS4 genes, it is possible that resistance to the pathogens is conferred by cooperation between the two NB-LRR genes.Several reports have shown that a single R gene/locus can confer resistance to multiple pathogens. For instance, tomato Mi mediates resistance against three distinct types of pests, including root-knot nematodes, potato aphids and sweet potato whitefly.¹⁰ In the present study, we suggest that two distinct R-genes located in a conserved head-to-head organization confer resistance to three distinct pathogen species by acting cooperatively.The tandem function of RRS1-Ws and its neighboring gene RPS4-Ws is also supported by the evolutionary conservation of the gene pair. Close homologs of RPS4 are often physically paired with homologs of RRS1 in a head-to-head (inverted) tandem arrangement.¹¹ The evolutionary conservation of homologous gene pairs in a head-to-head arrangement also supports the idea that cooperative function of two R genes could be a common mechanism of defense against pathogens. Since the two open reading frames are only 264 bp apart, the promoter regions of the gene pairs possibly overlap, leading to co-regulation of the genes. The head-to-head configuration may assure balanced levels of the protein pair to meet a strict stoichiometric requirement to act together, possibly in a complex. As a practical application, this finding may provide a new strategy for creating transgenic plants that express R genes from other plants. Introduction of two R genes in a head-to-head orientation may be necessary for effective pathogen resistance.     

Molecular cloning and gene expression of the prox1a and prox1b genes in the medaka,Oryzias latipes

Prox1 is a prospero-related homeobox gene. Prox1 is expressed in various internal organs and is related to those differentiations. Small fishes such as the zebrafish and the medaka are useful model animals in the clarification of the mechanism of development. The zebrafish prox1 is also identified, and it contributes to clarifying the function of prox1. However, it is necessary to note that many genes are duplicated in teleost fishes. In this study, we identified the orthologs of the mammalian prox1 gene in the medaka. The gene was also duplicated in the medaka, and we named it prox1a and prox1b. In silico analysis from the perspective of synteny indicated that medaka prox1a was similar to the prox1 gene of other vertebrates. Medaka prox1a was expressed in all internal organs that we have examined by RT-PCR. In contrast, medaka prox1b expression was limited to the brain, heart, liver, kidney, thymus, gill, testis, and ovary. This suggests that the two prox1 genes do not have a complementary relationship. In addition, we examined their expression patterns during embryonic development using whole-mount in situ hybridization. The expression pattern of prox1a showed a pattern similar to that of zebrafish prox1. In contrast, medaka prox1b was expressed asymmetrically in part of the central nervous system, especially strongly in the right side of the habenula.     

Tubulin polymerization inhibitors with a fluorinated phthalimide skeleton derived from thalidomide

4,7-Difluoro-2-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione [4,7FPP-33 (14)] has a potent tubulin-polymerization-inhibiting activity comparable with those of the known tubulin-polymerization inhibitors rhizoxin and colchicine. The structure-activity relationship for fluorine substitution was elucidated.     

Glycosylation engineering of therapeutic IgG antibodies: challenges for the safety,functionality and efficacy

Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic antibodies. While the biantennary complex-type oligosaccharide attached to Asn297 of the Fc is essential for antibody effector functions, fucose and outer-arm sugars attached to the core heptasaccharide that generate structural heterogeneity (glycoforms) exhibit unique biological activities. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for the development and quality control of therapeutic antibodies, and glycan profiles of the Fc are recognized as critical quality attributes. In the past decade our understanding of the influence of glycosylation on the structure/function of IgG-Fc has grown rapidly through X-ray crystallographic and nuclear magnetic resonance studies, which provides possibilities for the design of novel antibody therapeutics. Furthermore, the chemoenzymatic glycoengineering approach using endoglycosidase-based glycosynthases may facilitate the development of homogeneous IgG glycoforms with desirable functionality as nextgeneration therapeutic antibodies. Thus, the Fc glycans are fertile ground for the improvement of the safety, functionality, and efficacy of therapeutic IgG antibodies in the era of precision medicine.