Transformation: a tool for studying fungal pathogens of plants

Plant diseases caused by plant pathogenic fungi continuously threaten the sustainability of global crop production. An effective way to study the disease-causing mechanisms of these organisms is to disrupt their genes, in both a targeted and random manner, so as to isolate mutants exhibiting altered virulence. Although a number of techniques have been employed for such an analysis, those based on transformation are by far the most commonly used. In filamentous fungi, the introduction of DNA by transformation typically results in either the heterologous (illegitimate) integration or the homologous integration of the transforming DNA into the target genome. Homologous integration permits a targeted gene disruption by replacing the wild-type allele on the genome with a mutant allele on transforming DNA. This process has been widely used to determine the role of newly isolated fungal genes in pathogenicity. The heterologous integration of transforming DNA causes a random process of gene disruption (insertional mutagenesis) and has led to the isolation of many fungal mutants defective in pathogenicity. A big advantage of insertional mutagenesis over the more traditional chemical or radiation mutagenesis procedures is that the mutated gene is tagged by transforming DNA and can subsequently be cloned using the transforming DNA. The application of various transformation-based techniques for fungal gene manipulation and how they have increased our understanding and appreciation of some of the most serious plant pathogenic fungi are discussed. Received 9 May 2001; received after revision 2 July 2001; accepted 3 July 2001     

Binding of estradiol to synaptosomal mitochondria: physiological significance

The subsynaptosomal distribution and specific binding of 17beta-estradiol in vitro to mitochondria isolated from presynaptic nerve endings of female rat brain were examined. 17Beta-estradiol is (i) distributed unequally in synaptosomes and mitochondria posses the highest capacity to bind estradiol with respect to the available amount of the hormone. (ii) Estradiol binds specifically to isolated synaptosomal mitochondria. A Michaelis-Menten plot of specific binding was sigmoidal within a concentration range of 0.1-5 nM of added estradiol, with a saturation plateau at 3 nM. Binding of higher estradiol concentrations demonstrated an exponential Michaelis-Menten plot, indicating non-specific binding to mitochondria. Vmax and Km for the sigmoidal-shape range were estimated as 46 +/- 6 fmol of estradiol/mg of mitochondrial proteins and 0.46 +/- 0.07 nM free estradiol respectively. (iii) Estradiol binding is not affected by the removal of ovaries. The results show that inhibition of Na-dependent Ca2+ efflux from mitochondria by estradiol occurs according to an affinity change of the translocator for Na+, at the same estradiol concentrations that show specific binding to mitochondrial membranes. These data imply that physiological concentrations of estradiol, acting on mitochondrial membrane properties, extragenomically modulate the mitochondrial, and consequently the synaptosomal content of Ca2+, and in that way exert a significant change in nerve cell homeostasis.     

Neuronal plasticity and regeneration in the olfactory system of mammals: morphological and functional recovery following olfactory bulb deafferentation

The mammalian olfactory system has the unique property in the permanent turnover of the olfactory sensory neurons under normal conditions and following injury. This implies that the topographical map of the epithelium-to-bulb connections generated during ontogenesis has to be maintained despite neuron renewal in order to insure olfactory information processing. One way to investigate this issue has been to disrupt the peripheral connections and analyze how neural connections may be reestablished as well as how animals may perform in olfactory-mediated tasks. This review surveys the main data pertaining to both morphological and functional recoveries taking place in the peripheral olfactory system following olfactory bulb deafferentation. Conclusions from these studies are enlightened by recent data from molecular biology.     

Phylogeography of crossbills, bullfinches, grosbeaks, and rosefinches

Mitochondrial cytochrome b (cyt b) from 24 Carduelini species including crossbills, bullfinches, grosbeaks, rosefinches, and other related, but not conclusively classified species, was sequenced. These sequences were also compared with all the available sequences from the genera Carduelis, Serinus, and Passer. Phylogenetic analyses consistently gave the same groups of finches and the calculated divergence times suggest that speciation of the studied species occurred between 14 and 3 million years ago (Miocene-Pliocene), appearing before the Passer, Carduelis, and Serinus genera. Pleistocene glaciations may have been important in sub-speciation. Crossbills are integrated within the genus Carduelis, and within redpolls; the common crossbill shows subspeciation with Loxia japonica in the Pleistocene epoch. Pinicola enucleator groups together with bullfinches and is probably the ancestor of the group. Hawfinch is only distantly related to the studied groups, and might either represent an isolated genus or be related to the New World genus Hesperiphona. The grosbeak genera Eophona and Mycerobas are clearly sister groups, and species belonging to the former might have given rise to Mycerobas species. The isolated (in classification) Uragus sibiricus and Haematospiza sipahi are included within the genus Carpodacus (rosefinches); Carpodacus nipalensis is outside the genus Carpodacus in the molecular analyses and might be an isolated species or related to the genus Montifringilla.     

RGS2 regulates signal transduction in olfactory neurons by attenuating activation of adenylyl cyclase III

The heterotrimeric G-protein Gs couples cell-surface receptors to the activation of adenylyl cyclases and cyclic AMP production (reviewed in refs 1, 2). RGS proteins, which act as GTPase-activating proteins (GAPs) for the G-protein alpha-subunits alpha(i) and alpha(q), lack such activity for alpha(s) (refs 3-6). But several RGS proteins inhibit cAMP production by Gs-linked receptors. Here we report that RGS2 reduces cAMP production by odorant-stimulated olfactory epithelium membranes, in which the alpha(s) family member alpha(olf) links odorant receptors to adenylyl cyclase activation. Unexpectedly, RGS2 reduces odorant-elicited cAMP production, not by acting on alpha(olf) but by inhibiting the activity of adenylyl cyclase type III, the predominant adenylyl cyclase isoform in olfactory neurons. Furthermore, whole-cell voltage clamp recordings of odorant-stimulated olfactory neurons indicate that endogenous RGS2 negatively regulates odorant-evoked intracellular signalling. These results reveal a mechanism for controlling the activities of adenylyl cyclases, which probably contributes to the ability of olfactory neurons to discriminate odours.     

Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells

Natural killer (NK) cells destroy virus-infected and tumour cells, apparently without the need for previous antigen stimulation. In part, target cells are recognized by their diminished expression of major histocompatibility complex (MHC) class I molecules, which normally interact with inhibitory receptors on the NK cell surface. NK cells also express triggering receptors that are specific for non-MHC ligands; but the nature of the ligands recognized on target cells is undefined. NKp46 is thought to be the main activating receptor for human NK cells. Here we show that a soluble NKp46-immunoglobulin fusion protein binds to both the haemagglutinin of influenza virus and the haemagglutinin-neuraminidase of parainfluenza virus. In a substantial subset of NK cells, recognition by NKp46 is required to lyse cells expressing the corresponding viral glycoproteins. The binding requires the sialylation of NKp46 oligosaccharides, which is consistent with the known sialic binding capacity of the viral glycoproteins. These findings indicate how NKp46-expressing NK cells may recognize target cells infected by influenza or parainfluenza without the decreased expression of target-cell MHC class I protein.