Insecticide applications to soil contribute to the development of Burkholderia mediating insecticide resistance in stinkbugs

Some soil Burkholderia strains are capable of degrading the organophosphorus insecticide, fenitrothion, and establish symbiosis with stinkbugs, making the host insects fenitrothion‐resistant. However, the ecology of the symbiotic degrading Burkholderia adapting to fenitrothion in the free‐living environment is unknown. We hypothesized that fenitrothion applications affect the dynamics of fenitrothion‐degrading Burkholderia, thereby controlling the transmission of symbiotic degrading Burkholderia from the soil to stinkbugs. We investigated changes in the density and diversity of culturable Burkholderia (i.e. symbiotic and nonsymbiotic fenitrothion degraders and nondegraders) in fenitrothion‐treated soil using microcosms. During the incubation with five applications of pesticide, the density of the degraders increased from less than the detection limit to around 10⁶/g of soil. The number of dominant species among the degraders declined with the increasing density of degraders; eventually, one species predominated. This process can be explained according to the competitive exclusion principle using V_max and K_m values for fenitrothion metabolism by the degraders. We performed a phylogenetic analysis of representative strains isolated from the microcosms and evaluated their ability to establish symbiosis with the stinkbug Riptortus pedestris. The strains that established symbiosis with R. pedestris were assigned to a cluster including symbionts commonly isolated from stinkbugs. The strains outside the cluster could not necessarily associate with the host. The degraders in the cluster predominated during the initial phase of degrader dynamics in the soil. Therefore, only a few applications of fenitrothion could allow symbiotic degraders to associate with their hosts and may cause the emergence of symbiont‐mediated insecticide resistance.     

Protein expressed by the ho2 gene of the cyanobacterium Synechocystis sp. PCC 6803 is a true heme oxygenase. Properties of the heme and enzyme complex

Two isoforms of a heme oxygenase gene, ho1 and ho2, with 51% identity in amino acid sequence have been identified in the cyanobacterium Synechocystis sp. PCC 6803. Isoform-1, Syn HO-1, has been characterized, while isoform-2, Syn HO-2, has not. In this study, a full-length ho2 gene was cloned using synthetic DNA and Syn HO-2 was demonstrated to be highly expressed in Escherichia coli as a soluble, catalytically active protein. Like Syn HO-1, the purified Syn HO-2 bound hemin stoichiometrically to form a heme-enzyme complex and degraded heme to biliverdin IXalpha, CO and iron in the presence of reducing systems such as NADPH/ferredoxin reductase/ferredoxin and sodium ascorbate. The activity of Syn HO-2 was found to be comparable to that of Syn HO-1 by measuring the amount of bilirubin formed. In the reaction with hydrogen peroxide, Syn HO-2 converted heme to verdoheme. This shows that during the conversion of hemin to alpha-meso-hydroxyhemin, hydroperoxo species is the activated oxygen species as in other heme oxygenase reactions. The absorption spectrum of the hemin-Syn HO-2 complex at neutral pH showed a Soret band at 412 nm and two peaks at 540 nm and 575 nm, features observed in the hemin-Syn HO-1 complex at alkaline pH, suggesting that the major species of iron(III) heme iron at neutral pH is a hexa-coordinate low spin species. Electron paramagnetic resonance (EPR) revealed that the iron(III) complex was in dynamic equilibrium between low spin and high spin states, which might be caused by the hydrogen bonding interaction between the distal water ligand and distal helix components. These observations suggest that the structure of the heme pocket of the Syn HO-2 is different from that of Syn HO-1.     

TRAIL-transduced dendritic cells protect mice from acute graft-versus-host disease and leukemia relapse

TRAIL preferentially induces apoptotic cell death in a wide variety of transformed cells, whereas it induces no apoptosis, but inhibits activation of Ag-specific T cells via blockade of cell cycle progression. Although accumulating results suggest that TRAIL is involved in the maintenance of immunological homeostasis under steady state conditions as well as in the initiation and progression of immunopathologies, the potential regulatory effect of TRAIL on immune responses and its therapeutic potential in immunological diseases remains unclear. We report in this study the potential usefulness of TRAIL-transduced dendritic cells (DCs) for the treatment of lethal acute graft-vs-host disease (GVHD) and leukemia relapse. DCs genetically modified to express TRAIL showed potent cytotoxicity against both alloreactive T cells and leukemic cells through the induction of apoptosis. In addition, treatment with genetically modified DCs expressing TRAIL of allogeneic BM transplants recipients with leukemia was effective for protection against acute GVHD and leukemia relapse. Thus, gene transfer of TRAIL to DCs is a novel modality for the treatment of acute GVHD and leukemia relapse by selective targeting of pathogenic T cells and leukemic cells.     

An inducible RNA interference system in Physcomitrella patens reveals a dominant role of augmin in phragmoplast microtubule generation

Mitosis is a fundamental process of eukaryotic cell proliferation. However, the molecular mechanisms underlying mitosis remain poorly understood in plants partly because of the lack of an appropriate model cell system in which loss-of-function analyses can be easily combined with high-resolution microscopy. Here, we developed an inducible RNA interference (RNAi) system and three-dimensional time-lapse confocal microscopy in the moss Physcomitrella patens that allowed in-depth phenotype characterization of the moss genes essential for cell division. We applied this technique to two microtubule regulators, augmin and γ-tubulin complexes, whose mitotic roles remain obscure in plant cells. Live imaging of caulonemal cells showed that they proceed through mitosis with continual generation and self-organization of acentrosomal microtubules. We demonstrated that augmin plays an important role in γ-tubulin localization and microtubule generation from prometaphase to cytokinesis. Most evidently, microtubule formation in phragmoplasts was severely compromised after RNAi knockdown of an augmin subunit, leading to incomplete expansion of phragmoplasts and cytokinesis failure. Knockdown of the γ-tubulin complex affected microtubule formation throughout mitosis. We conclude that postanaphase microtubule generation is predominantly stimulated by the augmin/γ-tubulin machinery in moss and further propose that this RNAi system serves as a powerful tool to dissect the molecular mechanisms underlying mitosis in land plants.     

Interplay between MAMP-triggered and SA-mediated defense responses

Plants respond to pathogen infection using an innate immune system with at least two distinct recognition mechanisms. One mechanism recognizes microbe-associated molecular patterns (MAMPs). The other is based on resistance (R) genes and specifically recognizes certain pathogen virulence factors, including those delivered through the type III secretion system (TTSS) of bacteria. Salicylic acid (SA)-mediated responses are an important part of the R gene-mediated defense. Substantial overlaps between MAMP-triggered and SA-mediated responses have been reported. However, interactions between MAMP-triggered and SA-mediated signaling mechanisms have not been well documented. Here we report intimate interactions between MAMP-triggered and SA-mediated signaling. We found that SA accumulated at a higher level 6 h after treatment with a MAMP, flg22 or inoculation with Pseudomonas syringae pv. tomato DC3000 ( Pst DC3000) hrcC mutant, which is deficient in TTSS function. Disruptions of SA signaling components, such as SID2 and PAD4 , strongly affected MAMP-triggered responses monitored by expression profiling. We found two groups of genes that were induced by Pst DC3000 hrcC in an SA-dependent manner. One group was SID2 -dependent at all time points, whereas the other was SID2 -independent at early time points and SID2 -dependent at later time points. Thus, the expression of the latter genes responds to MAMPs through both SA-independent and SA-dependent signaling mechanisms. Strong resistance to Pst DC3000 hrcC was dependent on SA signaling. These results indicate that the SA increase triggered by MAMPs is a major component of the MAMP-triggered signaling mechanism, explaining the overlapping spectra of MAMP-triggered and SA-mediated responses.     

Enhanced activity of Mucor javanicus lipase in polyoxyethylene sorbitan trioleate containing microemulsion-based organogels

The efficacy of polyoxyethylene sorbitan trioleate (Tween 85) addition on the activity of Mucor javanicus lipase was investigated in sodium bis(2-ethylhexyl) sulfosuccinate (AOT) microemulsion-based organogels (MBGs). Gelatin was used as the gelling component of the MBGs. The maximal reaction rate was obtained at an AOT:Tween 85 molar ratio of 10:1. Under a fixed molar ratio system of AOT:Tween 85 = 10:1, the reaction rate also attained a maximum at a W_G (=[H₂O]/[AOT + Tween 85] in MBG phase) value of 100 and an AOT concentration of 150 mM. The reaction proceeded under a reaction-controlled regime, and the reaction rate for the AOT/Tween 85 mixed system was about 2-fold higher than that for the AOT single system. The lipase activity was well maintained for 10 days and recovered by contacting the MBGs with concentrated amphiphile solutions.