Control of volume-sensitive chloride channel inactivation by the coupled action of intracellular chloride and extracellular protons.

The volume-sensitive chloride current (I(ClVol)) exhibit a time-dependent decay presumably due to channel inactivation. In this work, we studied the effects of chloride ions (Cl(-)) and H(+) ions on I(ClVol) decay recorded in HEK-293 and HL-60 cells using the whole-cell patch clamp technique. Under control conditions ([Cl(-)](e) = [Cl(-)](i) = 140 mM and pH(i) = pH(e) = 7.3), I(ClVol) in HEK cells shows a large decay at positive voltages but in HL-60 cells I(ClVol) remained constant independently of time. In HEK-293 cells, simultaneously raising the [Cl(-)](e) and [Cl(-)](i) from 25 to 140 mM (with pH(e) = pH(i) = 7.3) increased the fraction of inactivated channels (FIC). This effect was reproduced by elevating [Cl(-)](i) while keeping the [Cl(-)](e) constant. Furthermore, a decrease in pH(e) from 7.3 to 5.5 accelerated current decay and increased FIC when [Cl(-)] was 140 mM but not 25 mM. In HL-60 cells, a slight I(ClVol) decay was seen when the pH(e) was reduced from 7.3 to 5.5. Our data show that inactivation of I(ClVol) can be controlled by changing either the Cl(-) or H(+) concentration or both. Based on our results and previously published data, we have built a model that explains VRAC inactivation. In the model the H(+) binding site is located outside the electrical field near the extracellular entry whilst the Cl(-) binding site is intracellular. The model depicts inactivation as a pore constriction that happens by simultaneous binding of H(+) and Cl(-) ions to the channel followed by a voltage-dependent conformational change that ultimately causes inactivation.

The MAP kinase TVK1 regulates conidiation, hydrophobicity and the expression of genes encoding cell wall proteins in the fungus Trichoderma virens.

Mitogen-activated protein (MAP) kinases modulate morphological and genetic processes, which determine cell fate. The tvk1 gene encodes a MAP kinase of Trichoderma virens and its deletion promotes an unusual conidiation phenotype in submerged culture. Here, it is reported that the morphology, physiology and expression of genes encoding cell wall proteins from Trichoderma are significantly affected by Tvk1. Morphological changes were evident in the cell walls of aerial conidia produced by a MAPK null mutant when compared to those produced by the wild-type. Unexpectedly, conidia produced in submerged culture by the Deltatvk1 strain were highly hydrophobic, whereas in aerial conidia hydrophobicity was severely reduced. In addition, the Deltatvk1 strain was unable to break the liquid-air interface when the fungus grew in rich medium; however, when it grew in minimal medium the fungus produced large filaments which were much more efficient at breaking the interface than the wild-type. Through cDNA subtractive hybridization between the wild-type and Deltatvk1 grown in submerged culture, five genes encoding hydrophobin-like proteins and two additional genes encoding cell wall proteins were identified. Four hydrophobin-encoding genes (Tv-hfb1, Tv-srh1, tv-cfth1 and Tv-qid3) and a gene encoding a clock-controlled-like protein (Tv-ccg14/TvSm1) were upregulated in Deltatvk1, whereas genes encoding a cell wall protein (tv-qid74) and an additional hydrophobin (tv-hfb3) were absent in the mutant strain. Clear differences in gene expression were shown during conidiation and emergence from the liquid-air interface, suggesting different functions of the corresponding proteins in these two phenomena. The results support a model in which Tvk1 regulates morphology and genes encoding cell wall proteins during development of Trichoderma.

Cross talk between a fungal blue-light perception system and the cyclic AMP signaling pathway.

Blue light regulates many physiological and developmental processes in fungi. In Trichoderma atroviride the complex formed by the BLR-1 and BLR-2 proteins appears to play an essential role as a sensor and transcriptional regulator in photoconidiation. Here we demonstrate that the BLR proteins are necessary for carbon deprivation induced conidiation, even in the absence of light, pointing to the existence of an unprecedented cross talk between light and carbon sensing. Further, in contrast to what has been found in all other fungal systems, clear BLR-independent blue-light responses, including the activation of protein kinase A (PKA) and the regulation of gene expression, were found. Expression of an antisense version of the pkr-1 gene, encoding the regulatory subunit of PKA, resulted in a nonsporulating phenotype, whereas overexpression of the gene produced colonies that conidiate even in the dark. In addition, overexpression of pkr-1 blocked the induction of early light response genes. Thus, our data demonstrate that PKA plays an important role in the regulation of light responses in Trichoderma. Together, these observations suggest that the BLR complex plays a general role in sensing environmental cues that trigger conidiation and that such a role can be separated from its function as a transcription factor.

Three decades of fungal transformation: novel technologies.

Fungi are lower eukaryotes that play important roles in many human activities, including biotechnological processes, phytopathology, and biomedical research. In addition, they are excellent models for molecular and genetic studies. An important key in the advancement of genetics and molecular biology of a given organism is the development of genetic transformation systems. This technology makes possible the analysis and manipulation of the genome of the organism of interest. Thirty years from the first report of transformation of a fungus, transformation of many other fungi has been achieved. However, the development of gene tagging systems generally applicable to a wide range of filamentous fungi has remained elusive. A widely used gene tagging strategy for filamentous fungi is restriction enzyme mediated integration (REMI). In recent years numerous reports have been published describing the effective application of REMI. However, REMI shows certain disadvantages for some fungi. Recently a very promising alternative strategy has been reported based on the use of the soil bacterium Agrobacterium tumefaciens. Using this system a well-defined DNA segment (T-DNA) is transferred, which integrates by illegitimate recombination and is 100-1000 times more efficient than conventional methods. The T-DNA can be used as an efficient tool to generate recombinant strains where DNA is integrated as a single copy, allowing the generation of collections of gene-tagged mutants of the fungus of interest.