Selected isolates of Trichoderma gamsii induce different pathways of systemic resistance in maize upon Fusarium verticillioides challenge.

The pink ear rot is one of the most damaging maize diseases, caused by the mycotoxigenic fungal pathogen, Fusarium verticillioides. The application of biological control agents, like antagonistic and/or resistance inducer microorganisms, is an option to reduce fungal infection and kernel contamination in a sustainable and environmentally friendly way. It is well known that Trichoderma species are non-pathogenic fungi able to antagonize plant pathogens and to induce systemic resistance in plants. The present work aimed to verify if Trichoderma spp., applied to maize kernels, affect the plant growth and induce systemic responses to F. verticillioides. Besides, the capability to reduce fumonisin concentration in liquid cultures was investigated. Two T. gamsii (IMO5 and B21), and one T. afroharzianum (B75) isolates, selected both for antagonism and for the ability to reduce root infections, significantly reduced the endophytic development of the stem-inoculated pathogen, compared to the control. The mechanisms of action appeared to be strain-specific, with IMO5 enhancing transcript levels of marker genes of Induced Systemic Resistance (ZmLOX10, ZmAOS, and ZmHPL) while B21 enhancing marker genes of Systemic Acquired Resistance (ZmPR1 and ZmPR5), as evinced by measuring their expression profiles in the leaves. Moreover, IMO5 promoted plant growth, while B21 was able to significantly reduce the fumonisin content in a liquid medium. The results of this work give new evidence that the seed application of T. gamsii is a promising tool for controlling F. verticillioides to be integrated with breeding and the adoption of good agricultural practices.

Microplate-Based Evaluation of the Sugar Yield from Giant Reed, Giant Miscanthus and Switchgrass after Mild Chemical Pre-Treatments and Hydrolysis with Tailored Trichoderma Enzymatic Blends.

Giant reed, miscanthus, and switchgrass are considered prominent lignocellulosic feedstocks to obtain fermentable sugars for biofuel production. The bioconversion into sugars requires a delignifying pre-treatment step followed by hydrolysis with cellulase and other accessory enzymes like xylanase, especially in the case of alkali pre-treatments, which retain the hemicellulose fraction. Blends richer in accessory enzymes than commercial mix can be obtained growing fungi on feedstock-based substrates, thus ten selected Trichoderma isolates, including the hypercellulolytic strain Trichoderma reesei Rut-C30, were grown on giant reed, miscanthus, or switchgrass-based substrates. The produced enzymes were used to saccharify the corresponding feedstocks, compared to a commercial enzymatic mix (6 FPU/g). Feedstocks were acid (H2SO4 0.2-2%, w/v) or alkali (NaOH 0.02-0.2%, w/v) pre-treated. A microplate-based approach was chosen for most of the experimental steps due to the large number of samples. The highest bioconversion was generally obtained with Trichoderma harzianum Or4/99 enzymes (78, 89, and 94% final sugar yields at 48 h for giant reed, miscanthus, and switchgrass, respectively), with significant increases compared to the commercial mix, especially with alkaline pre-treatments. The differences in bioconversion yields were only partially caused by xylanases (maximum R 2 = 0.5), indicating a role for other accessory enzymes.

Isatis canescens is a rich source of glucobrassicin and other health-promoting compounds.

BACKGROUND: Glucobrassicin (GBS), a glucosinolate contained in many brassica vegetables, is the precursor of chemopreventive compounds such as indole-3-carbinol. Large amounts of GBS would be needed to perform studies aimed at elucidating its role in the diet. This study was mainly undertaken to evaluate the flower buds of Isatis canescens as a source for GBS purification. In order to investigate the health-promoting potential of this species, glucosinolate, phenol and flavonoid content as well as the whole antioxidant capacity were also determined. Flower bud samples were collected in four localities around Mount Etna in Sicily, Italy, where I. canescens is widespread, as they are locally traditionally eaten. RESULTS: I. canescens flower buds displayed high GBS concentrations, up to 60 µmol g(-1) dry weight. The purification method consisted of two chromatographic steps, which made it possible to obtain GBS with a purity of 92-95%, with a yield of 21 g kg(-1) . The total glucosinolates, phenols, flavonoids and antioxidant activity were considerable, with the southern locality showing the highest concentrations for all the phytochemicals. CONCLUSION: I. canescens flower buds represent a naturally rich source of GBS, at a level suitable for its purification. Furthermore, flower bud consumption could provide an intake of health-promoting compounds, with possible antioxidant and chemopreventive properties.

Evaluation of selected white-rot fungal isolates for improving the sugar yield from wheat straw.

Biological pretreatment of lignocellulosic biomass by fungi can represent a low-cost and eco-friendly alternative to physicochemical methods to facilitate enzymatic hydrolysis. However, fungal metabolism can cause cellulose loss and it is therefore necessary to use the appropriate fungal strain-biomass type combination. In this work, the effects of biological pretreatments carried out by five different fungi on enzymatic hydrolysis of wheat straw were investigated. The best results were obtained with a Ceriporiopsis subvermispora strain, which minimized weight and cellulose losses and gave the highest net sugar yield (calculated with respect to the holocellulose content of the untreated straw), up to 44 % after a 10-week pretreatment, more than doubling the yields obtained with the other isolates. Moreover, prolonging the pretreatment from 4 up to 10 weeks produced a 2-fold increase, up to 60 %, in digestibility (sugar yield, calculated considering the holocellulose content of the pretreated material). The hemicellulose content of the pretreated material resulted inversely correlated with digestibility, and it could thus be utilized as an index of the pretreatment efficacy. Finally, a correlation was also found between digestibility and the difference between the absorbance values at 290 and 320 nm of pretreated wheat straw extracts.

Hydrolytic potential of Trichoderma sp. strains evaluated by microplate-based screening followed by switchgrass saccharification.

Bioconversion of lignocellulosic biomass to fuel requires a hydrolysis step to obtain fermentable sugars, generally accomplished by fungal enzymes. Large-scale screening of different microbial strains would provide optimal enzyme cocktails for any target feedstock. The aim of this study was to screen a large collection of Trichoderma sp. strains for the hydrolytic potential towards switchgrass (Panicum virgatum L.). Strains were cultivated in a small-scale system and assayed in micro-plates for xylanase and cellulase activities. The population distributions of these traits are reported after growth on switchgrass in comparison with cellulose. The distribution profiles suggest that the growth on switchgrass strongly promotes xylanase production. The IK4 strain displayed the highest xylanase activity after growth on switchgrass (133U/mL). Enzymes (10FPU/g substrate) from IK4 were compared with those from 2 cellulolytic Trichoderma strains and a commercial enzyme in saccharification time-course experiments on untreated and pretreated switchgrass and on an artificial substrate. Samples were analysed by DNS assay and by an oxygraphic method for sugar equivalent or glucose concentration. On the untreated substrate, IK4 enzymes even outperformed a 5-fold load of commercial enzyme, suggesting that xylanase or accessory enzymes are a limiting factor on this type of recalcitrant substrate. On the other substrates, IK4 preparations showed intermediate behaviour if compared with the commercial enzyme at 10FPU/g substrate and at 5-fold load. IK4 also nearly halved the time to release 50% of the hydrolysable sugar equivalents (T(50%)), with respect to the other preparations at the same enzymatic load. DNS assay and oxygraphic method gave highly correlated results for the 3 saccharified substrates. The study suggests that accessory enzymes like xylanase play a key role in improving the performance of cellulase preparations on herbaceous lignocellulosic feedstocks like switchgrass.

A novel microplate-based screening strategy to assess the cellulolytic potential of Trichoderma strains.

Bioconversion of lignocellulosic biomass to fuel requires a hydrolysis step to obtain fermentable sugars, generally accomplished by fungal enzymes. An assorted library of cellulolytic microbial strains should facilitate the development of optimal enzyme cocktails specific for locally available feedstocks. Only a limited number of strains can be simultaneously assayed in screening based on large volume cultivation methods, as in shake flasks. This study describes a miniaturization strategy aimed at allowing parallel assessment of large numbers of fungal strains. Trichoderma strains were cultivated stationary on microcrystalline cellulose using flat bottom 24-well plates containing an agarized medium. Supernatants obtained by a rapid centrifugation step of the whole culture plates were evaluated for extracellular total cellulase activity, measured as filter paper activity, using a microplate-based assay. The results obtained were consistent with those observed in shake-flask experiments and more than 300 Trichoderma strains were accordingly characterized for cellulase production. Five strains, displaying on shake-flasks at least 80% of the activity shown by the hyper-cellulolytic mutant Trichoderma Rut-C30, were correctly recognized by the screening on 24-well plates, demonstrating the feasibility of this approach. Cellulase activity distribution for the entire Trichoderma collection is also reported. One strain (T. harzianum Ba8/86) displayed the closest profile to the reference strain Rut-C30 in time course experiments. The method is scalable and addresses a major bottleneck in screening programs, allowing small-scale parallel cultivation and rapid supernatant extraction. It can also be easily integrated with high-throughput enzyme assays and could be suitable for automation.

Perchlorate transport and inhibition of the sodium iodide symporter measured with the yellow fluorescent protein variant YFP-H148Q/I152L.

Perchlorate is an environmental contaminant that impairs thyroid function by interacting with the sodium iodide symporter (NIS), the transporter responsible for iodide uptake in the thyroid gland. Perchlorate is well known as a competitive inhibitor of iodide transport by NIS, and recent evidence demonstrates that NIS can also transport perchlorate. In this study, we evaluated the yellow fluorescent protein (YFP) variant YFP-H148Q/I152L, as a genetically encodable biosensor of intracellular perchlorate concentration monitored by real-time fluorescence microscopy. Fluorescence of recombinant YFP-H148Q/I152L was suppressed by perchlorate and iodide with similar affinities of 1.2 mM and 1.6 mM, respectively. Perchlorate suppressed YFP-H148Q/I152L fluorescence in FRTL-5 thyroid cells and NIS-expressing COS-7 cells, but had no effect on COS-7 cells lacking NIS. Fluorescence changes in FRTL-5 cells were Na+-dependent, consistent with the Na+-dependence of NIS activity. Perchlorate uptake in FRTL-5 cells resulted in 10-fold lower intracellular concentrations than iodide uptake, and was characterized by a higher affinity (K(m) 4.6 microM for perchlorate and 34.8 muM for iodide) and lower maximal velocity (V(max) 6.8 microM/s for perchlorate and 39.5 microM/s for iodide). Perchlorate also prevented iodide-induced changes in YFP-H148Q/I152L fluorescence in FRTL-5 cells, with half-maximal inhibition occurring at 1.1-1.6 muM. In conclusion, YFP-H148Q/I152L detects perchlorate accumulation by thyroid and other NIS-expressing cells, and reveals differences in the kinetics of perchlorate versus iodide transport by NIS.

Proteolysis of the proofreading subunit controls the assembly of Escherichia coli DNA polymerase III catalytic core.

The C-terminal region of the proofreading subunit (epsilon) of Escherichia coli DNA polymerase III is shown here to be labile and to contain the residues (identified between F187 and R213) responsible for association with the polymerase subunit (alpha). We also identify two alpha-helices of the polymerase subunit (comprising the residues E311-M335 and G339-D353, respectively) as the determinants of binding to epsilon. The C-terminal region of epsilon is degraded by the ClpP protease assisted by the GroL molecular chaperone, while other factors control the overall concentration in vivo of epsilon. Among these factors, the chaperone DnaK is of primary importance for preserving the integrity of epsilon. Remarkably, inactivation of DnaK confers to Escherichia coli inviable phenotype at 42 degrees C, and viability can be restored over-expressing epsilon. Altogether, our observations indicate that the association between epsilon and alpha subunits of DNA polymerase III depends on small portions of both proteins, the association of which is controlled by proteolysis of epsilon. Accordingly, the factors catalysing (ClpP, GroL) or preventing (DnaK) this proteolysis exert a crucial checkpoint of the assembly of Escherichia coli DNA polymerase III core.

Fluorescence quantitation of thyrocyte iodide accumulation with the yellow fluorescent protein variant YFP-H148Q/I152L.

The thyroid gland accumulates iodide for the synthesis of thyroid hormones. The aim of the current study was to quantify iodide accumulation in cultured thyroid cells by live cell imaging using the halide-sensitive yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In vivo calibrations were performed in FRTL-5 thyrocytes to determine the sensitivity of YFP-H148Q/I152L to iodide. In the presence of ion-selective ionophores, YFP-H148Q/I152L fluorescence was suppressed by halides in a pH-dependent manner with 20-fold selectivity for iodide versus chloride and competition between the two halides. At a physiological pH of 7 and a chloride concentration of 15mM, the affinity constant of YFP-H148Q/I152L for iodide was 3.5mM. In intact FRTL-5 cells, iodide induced a reversible decrease in YFP-H148Q/I152L fluorescence. FRTL-5 cells concentrated iodide to 60 times the extracellular concentration. Iodide influx exhibited saturation kinetics with respect to extracellular iodide with a K(m) of 35 microM and a V(max) of 55 microM/s. Iodide efflux exhibited saturation kinetics with respect to intracellular iodide concentration with a K(m) of 2.2mM and a V(max) of 43 microM/s. The results of this study demonstrate the utility of YFP-H148Q/I152L as a sensitive and selective biosensor for the quantification of iodide accumulation in thyroid cells.

Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L.

The sodium iodide symporter (NIS) mediates iodide (I(-)) transport in the thyroid gland and other tissues and is of increasing importance as a therapeutic target and nuclear imaging reporter. NIS activity in vitro is currently measured with radiotracers and electrophysiological techniques. We report on the development of a novel live cell imaging assay of NIS activity using the I(-)-sensitive and genetically encodable yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In FRTL-5 thyrocytes stably expressing YFP-H148Q/I152L, I(-) induced a rapid and reversible decrease in cellular fluorescence characterized by 1) high affinity for extracellular I(-) (35 muM), 2) inhibition by the NIS inhibitor perchlorate, 3) extracellular Na(+) dependence, and 4) TSH dependence, suggesting that fluorescence changes are due to I(-) influx via NIS. Individual cells within a population of FRTL-5 cells exhibited a 3.5-fold variation in the rate of NIS-mediated I(-) influx, illustrating the utility of YFP-H148Q/I152L to detect cell-to-cell difference in NIS activity. I(-) also caused a perchlorate-sensitive decrease in YFP-H148Q/I152L fluorescence in COS-7 cells expressing NIS but not in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of NIS-mediated I(-) uptake in thyroid cells and in nonthyroidal cells following gene transfer and suggest that fluorescence detection of cellular I(-) may be a useful tool by which to study the pathophysiology and pharmacology of NIS.

Silencing of the gene coding for the epsilon subunit of DNA polymerase III slows down the growth rate of Escherichia coli populations.

Chromosome replication in Escherichia coli is accomplished by the multimeric enzyme DNA polymerase III; the relevance, in vivo, of the epsilon subunit (encoded by dnaQ) for processivity and fidelity of DNA polymerase III has been evaluated. To this aim, dnaQ has been conditionally silenced by means of in vivo expression of different antisense RNAs. Unexpectedly, the presence of the Shine-Dalgarno sequence is essential for the effectiveness of antisense constructs. Silencing of dnaQ induces a severe decrease in growth rate not paralleled by high mutation frequencies, suggesting that the epsilon subunit primarily affects the processivity of DNA polymerase III.