Effects of actinobacteria on plant disease suppression and growth promotion

Biological control and plant growth promotion by plant beneficial microbes has been viewed as an alternative to the use of chemical pesticides and fertilizers. Bacteria and fungi that are naturally associated with plants and have a beneficial effect on plant growth by the alleviation of biotic and abiotic stresses were isolated and developed into biocontrol (BCA) and plant growth-promoting agents (PGPA). Actinobacteria are a group of important plant-associated spore-forming bacteria, which have been studied for their biocontrol, plant growth promotion, and interaction with plants. This review summarizes the effects of actinobacteria as BCA, PGPA, and its beneficial associations with plants.     

Promoter-trapping in Saccharomyces cerevisiae by radiation-assisted fragment insertion

Non-homologous insertion (NHI) of DNA fragments into genomic DNA is a method widely used in insertional mutagenesis screens. In the yeast Saccharomyces cerevisiae, the efficiency of NHI is very low. Here we report that its efficiency can be increased by γ-irradiation of recipient cells at the time of transformation. Radiation-assisted NHI depends on YKU70, but its efficiency is not improved by inactivation of RAD5 or RAD52. In a pilot study, we generated 102 transformant clones expressing a lacZ reporter gene under standard conditions (30°C, rich medium). The site of insertion was determined in a subset of eight clones in which lacZ expression was altered by UV-irradiation. A comparison with published data revealed that three of the eight genes identified in our screen have not been targeted by large-scale transposon-based insertion screens. This suggests that radiation-assisted NHI offers a more homogeneous coverage of the genome than methods relying on transposons or retroviral elements.     

Infection of H69AR cells with retroviral particles harboring interfering RNAi significantly reduced the multidrug resistance of these small cell lung cancer cells

Incubation of the drug-sensitive H69, a small cell lung cancer cell line, with increased concentrations of adriamycin yielded multidrug resistant (MDR) H69AR cells that over-express multidrug resistance-associated protein (MRP1). MRP1 co-transports its substrate with glutathione (GSH), leading to lower intracellular GSH. In this report we tested whether depleting intracellular GSH in MRP1-expressing cells could hyper-sensitize them to anticancer drugs or not. We have found that the GSH contents in MRP1-expressing cells are significantly lower than their corresponding control cells. The treatment with MRP1 substrate verapamil or the GSH synthetase inhibitor buthionine sulfoxi-mine significantly reduced the intracellular GSH contents in MRP1-expressing cells. Interestingly, depleting intracellular GSH contents can hyper-sensitize the MRP1-cDNA transfected BHK cells to daunomycin, but not the adriamycin-selected H69AR cells. Further analyses indicated that anti-apoptotic factor Bcl2 might be a factor responsible for the fact that depleting intracellular GSH could not hyper-sensitize H69AR cells to daunomycin. We hypothesized that knocking down the expression of Bcl2 could hyper-sensitize H69AR cells to daunomycin. Interestingly, infection of H69AR cells with retroviral particles harboring Bcl2 interfering RNAi not only reduced the expression of Bcl2, but also many factors that contribute to MDR, such as Bcl-xl, MRP1 and ABCC3, etc., leading to the MDR H69AR cells more sensitive to daunomycin than the parental H69 cell. Thus, although the mechanisms of the down-regulation of the genes contributing to MDR remain to be elucidated, retroviral particles harboring Bcl2 interfering RNAi could be used as an alternative way to sensitize the MDR cancer cells to anticancer drugs.     

Beneficial effects of edaravone, a novel antioxidant, in rats with dilated cardiomyopathy

Edaravone, a novel antioxidant, acts by trapping hydroxyl radicals, quenching active oxygen and so on. Its cardioprotective activity against experimental autoimmune myocarditis (EAM) was reported. Nevertheless, it remains to be determined whether edaravone protects against cardiac remodelling in dilated cardiomyopathy (DCM). The present study was undertaken to assess whether edaravone attenuates myocardial fibrosis, and examine the effect of edaravone on cardiac function in rats with DCM after EAM. Rat model of EAM was prepared by injection with porcine cardiac myosin 28 days after immunization, we administered edaravone intraperitoneally at 3 and 10 mg/kg/day to rats for 28 days. The results were compared with vehicle-treated rats with DCM. Cardiac function, by haemodynamic and echocardiographic study and histopathology were performed. Left ventricular (LV) expression of NADPH oxidase subunits (p47(phox), p67(phox), gp91(phox) and Nox4), fibrosis markers (TGF-β(1) and OPN), endoplasmic reticulum (ER) stress markers (GRP78 and GADD 153) and apoptosis markers (cytochrome C and caspase-3) were measured by Western blotting. Edaravone-treated DCM rats showed better cardiac function compared with those of the vehicle-treated rats. In addition, LV expressions of NADPH oxidase subunits levels were significantly down-regulated in edaravone-treated rats. Furthermore, the number of collagen-III positive cells in the myocardium of edaravone-treated rats was lower compared with those of the vehicle-treated rats. Our results suggest that edaravone ameliorated the progression of DCM by modulating oxidative and ER stress-mediated myocardial apoptosis and fibrosis.     

Expression analysis on mycoparasitism related genes during antagonism of <Emphasis Type="Italic">Trichoderma</Emphasis> with <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis> causing red rot in sugarcane

Present study was aimed to select a suitable Trichoderma isolate as candidate antagonist based on its efficacy in producing cell wall degrading enzymes (CWDEs), its mycoparasitism activity and expression of related genes against the red rot pathogen caused by Colletotrichum falcatum in sugarcane. For which, six different isolates of Trichoderma selected from our earlier studies (T. harzianum, T. asperullum) were evaluated based on their capability in releasing cell wall degrading enzymes individually and during antagonism with C. falcatum in dual plate. Amongst T. harzianum (T20) exhibited the greatest mycoparasitic potential against the C. falcatum, by producing higher concentration of  CWDEs viz., chitinase and β-1, 3-glucanase, slightly lower amounts of cellulase and protease with significant reduction in polygalacturonase produced by pathogen. Further microscopic observation on interaction of C. falcatum with the selected isolate of T. harzianum (T20) exhibited the mycoparasitic activity of antagonist over pathogen in dual culture and inhibition of C. falcatum pathogenesis in detached sugarcane leaves. In addition, expression pattern of eight genes coding various enzymes involved in mycoparasitism by T. harzianum over C. falcatum were analyzed using qRT-PCR in vitro and on sugarcane leaves. In in vitro interactions, five genes of  cell wall degrading enzymes viz., chitinase (chit33), endochitinase (endo42), β-1, 3-glucanase (glu), exochitinase 1 (exc1), exochitinase 2 (exc2), were upregulated during and after contact as compared to before contact, while three genes related with proteases such as alkaline proteinase (prb1), trypsin-like protease (Pra1), subtilin-like serine protease (ssp), genes were upregulated during the contact with C. falcatum and slightly down regulated after contact. In detached leaves, seven genes were potentially upregulated except subtilin-like serine protease, which was down regulated during interaction of C. falcatum and T. harzianum as compared to T. harzianum inoculation alone. All these biochemical and molecular results confirm the efficacy of T. harzianum (T20) against C. falcatum and justify the right selection of candidate antagonist for our further studies on identification of antifungal genes/proteins against C. falcatum in sugarcane.     

In Vitro Characterization of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strains with Inhibitory Activity on Enteropathogens for Use as Potential Animal Probiotics

The present study evaluates the probiotic properties of three Lactobacillus plantarum strains MJM60319, MJM60298, and MJM60399 possessing antimicrobial activity against animal enteric pathogens. The three strains did not show bioamine production, mucinolytic and hemolytic activity and were susceptible to common antibiotics. The L. plantarum strains survived well in the simulated orogastrointestinal transit condition and showed adherence to Caco-2 cells in vitro. The L. plantarum strains showed strong antimicrobial activity against enterotoxigenic Escherichia coli, Shiga toxin-producing E. coli, Salmonella enterica subsp. enterica serovar Typhimurium, Choleraesuis and Gallinarum compared to the commercial probiotic strain Lactobacillus rhamnosus GG. The mechanism of antimicrobial activity of the L. plantarum strains appeared to be by the production of lactic acid. Furthermore, the L. plantarum strains tolerated freeze-drying and maintained higher viability in the presence of cryoprotectants than without cryoprotectants. Finally, the three L. plantarum strains tolerated NaCl up to 8% and maintained >60% growth. These characteristics of the three L. plantarum strains indicate that they could be applied as animal probiotic after appropriate in vivo studies.     

Glutamine residues in Q-loops of multidrug resistance protein MRP1 contribute to ATP binding via interaction with metal cofactor

Structural analyses of bacterial ATP-binding-cassette transporters revealed that the glutamine residue in Q-loop plays roles in interacting with: 1) a metal cofactor to participate in ATP binding; 2) a putative catalytic water molecule to participate in ATP hydrolysis; 3) other residues to transmit the conformational changes between nucleotide-binding-domains and transmembrane-domains, in ATP-dependent solute transport. We have mutated the glutamines at 713 and 1375 to asparagine, methionine or leucine to determine the functional roles of these residues in Q-loops of MRP1. All these single mutants significantly decreased Mg·ATP binding and increased the K(m) (Mg·ATP) and V(max) values in Mg·ATP-dependent leukotriene-C4 transport. However, the V(max) values of the double mutants Q713N/Q1375N, Q713M/Q1375M and Q713L/Q1375L were lower than that of wtMRP1, implying that the double mutants cannot efficiently bind Mg·ATP. Interestingly, MRP1 has higher affinity for Mn·ATP than for Mg·ATP and the Mn·ATP-dependent leukotriene-C4 transport activities of Q713N/Q1375N and Q713M/Q1375M are significantly higher than that of wtMRP1. All these results suggest that: 1) the glutamine residues in Q-loops contribute to ATP-binding via interaction with a metal cofactor; 2) it is most unlikely that these glutamine residues would play crucial roles in ATP hydrolysis and in transmitting the conformational changes between nucleotide-binding-domains and transmembrane-domains.