Coupling cell division and cell death to microtubule dynamics

The mitotic spindle is a self-organizing structure that is constructed primarily from microtubules. Among the most important spindle microtubules are those that bind to kinetochores and form the fibers along which chromosomes move. Chemotherapeutics such as taxol and the vinca alkaloids perturb kinetochore—microtubule attachment and disrupt chromosome segregation. This activates a checkpoint pathway that delays cell cycle progression and induces programmed cell death. Recent work has identified at least four mammalian spindle assembly checkpoint proteins.     

Two-component signal transduction systems in eukaryotic microorganisms

Conserved signal transduction pathways that use phosphorelay from histidine kinases through an intermediate transfer protein (H2) to response regulators have been found in a variety of eukaryotic microorganisms. Several of these pathways are linked to mitogen-activated protein kinase cascades. These networks control different physiological responses including osmoregulation, cAMP levels and cellular morphogenesis.     

Dimorphism and virulence in Candida albicans

Two regulatory pathways govern filamentation in the pathogenic fungus Candida albicans. Recent virulence studies of filamentation regulatory mutants argue that both yeast and filamentous forms have roles in infection. Filamentation control pathways seem closely related in C. albicans and in Saccharomyces cerevisiae, thus permitting speculation about C. albicans filamentation genes not yet discovered.     

稻瘟菌无毒基因研究进展

稻瘟菌是引起水稻稻瘟病的病原物。水稻与稻瘟菌间存在广泛而特异的相互作用,是研究寄主与病原物互作的重要模式系统。本文对稻瘟菌与水稻互作最重要的激发子―无毒基因的研究现状进行了概括,讨论了无毒基因的定位、克隆方法以及已克隆无毒基因的功能及进化研究,同时对今后无毒基因研究的重要方向进行了探讨,为深入理解无毒基因的功能及与水稻可能的互作关系奠定了基础。     

Transformation of the rice blast fungus Magnaporthe grisea to benomyl resistance

A transformation method based on a dominant selectable marker (benomyl resistance) was developed for the rice blast fungus Magnaporthe grisea. The heterologous gene for -tubulin from Neurospora crassa (pBT3) was used to obtain benomyl-resistant M. grisea transformants at a frequency of 20 to 30/g of DNA. Control transformations carried out with a plasmid conferring hygromycin resistance or a derivative of pBT3 containing a repetitive DNA sequence, yielded the same frequency of transformation as that of pBT3. Molecular analysis of the transformants indicated multiple integration of the vector DNA.     

Infection-related development in the rice blast fungus Magnaporthe grisea

Recent developments have been made in the identification of signal transduction pathways and gene products involved in the infection-related development of the rice blast fungus, Magnaporthe grisea. It has been established that cAMP-dependent and MAP kinase-mediated signaling are both critical for appressorium morphogenesis and function. These signaling pathways may act downstream of hydrophobin-mediated surface sensing by the growing germ tube. Several genes have been identified that are required for invasive growth of M. grisea including genes that allow adaptation of fungal metabolism to growth within plant tissues.     

LOX genes in blast fungus (Magnaporthe grisea) resistance in rice

Plant Lipoxygenases (LOX) are known to play major role in plant immunity by providing front-line defense against pathogen-induced injury. To verify this, we isolated a full-length OsLOX3 gene and also 12 OsLOX cDNA clones from Oryza sativa indica (cultivar Pusa Basmati 1). We have examined the role played by LOXs in plant development and during attack by blast pathogen Magnaporthe grisea. Gene expression, promoter region analysis, and biochemical and protein structure analysis of isolated OsLOX3 revealed significant homology with LOX super family. Protein sequence comparison of OsLOXs revealed high levels of homology when compared with japonica rice (up to100%) and Arabidopsis (up to 64%). Isolated LOX3 gene and 12 OsLOX cDNAs contained the catalytic LOX domains much required for oxygen binding and synthesis of oxylipins. Amino acid composition, protein secondary structure, and promoter region analysis (with abundance of motifs CGTCA and TGACG) support the role of OsLOX3 gene in providing resistance to diseases in rice plants. OsLOX3 gene expression analysis of root, shoot, flag leaf, and developing and mature seed revealed organ specific patterns during rice plant development and gave evidence to association between tissue location and physiological roles played by individual OsLOXs. Increased defense activity of oxylipins was observed as demonstrated by PCR amplification of OsLOX3 gene and upon inoculation with virulent strains of M. grisea and ectopic application of methyl jasmonate in the injured leaf tissue in adult rice plants.     

Cellular differentiation and host invasion by the rice blast fungus Magnaporthe grisea

This review describes current advances in understanding the biology of plant infection by the rice blast fungus Magnaporthe grisea. Development of the specialized infection structure, the appressorium, in M. grisea has recently been shown to be controlled by cell cycle progression and initiation of autophagic, programmed cell death in the fungal spore. Re-cycling of the contents of the fungal spore and peroxisomal fatty acid beta-oxidation are therefore important processes for appressorium function. Following entry to the host plant, new evidence suggests that M. grisea grows biotrophically within rice cells, bounded by the plant plasmalemma, and the fungus moves from cell-to-cell by means of plasmodesmata. Biotrophic proliferation of the fungus is likely to require secretion of effector proteins and suppression of host defences. Consistent with this, a component of the polarized exocytosis machinery of M. grisea is necessary for pathogenicity and also for induction of host defences in an incompatible interaction. Large-scale insertional mutagenesis is now allowing the rapid analysis of gene function in M. grisea, heralding a new approach to the study of this important fungal pathogen.     

Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice

Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects. Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthe grisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.     

Microsatellite markers for population studies of the rice blast fungus,Magnaporthe grisea

We developed nine new microsatellite markers for rice blast (Magnaporthe grisea) population studies. These markers were used in addition to nine microsatellite markers previously developed by our group for mapping purpose. Altogether, the 18 markers were used in multiplex PCR (polymerase chain reaction) to characterize six populations from different geographical origins. The average number of alleles per locus across populations ranged from 1.2 to 7 and the total number of alleles detected from 2 to 19. Based on this large range of polymorphism, this set of markers is expected to be useful for different kind of population studies at different geographical scales.     

Regulation of the MPG1 hydrophobin gene in the rice blast fungus Magnaporthe grisea

The hydrophobin-encoding gene MPG1 of the rice blast fungus Magnaporthe grisea is highly expressed during the initial stages of host plant infection and targeted deletion of the gene results in a mutant strain that is reduced in virulence, conidiation, and appressorium formation. The green fluorescent protein-encoding allele sGFP was used as a reporter to investigate regulatory genes that control MPG1 expression. The MAP kinase-encoding gene PMK1 and the wide domain regulators of nitrogen source utilization, NPR1 and NUT1, were required for full expression of MPG1 in response to starvation stress. The CPKA gene, encoding the catalytic subunit of protein kinase A, was required for repression of MPG1 during growth in rich nutrient conditions. During appressorium morphogenesis, high-level MPG1 expression was found to require the CPKA and NPR1 genes. Expression of a destabilized GFP allele indicated that de novo MPG1 expression occurs during appressorium formation. Three regions of the MPG1 promoter were identified which are required for high-level expression of MPG1 during appressorium formation and are necessary for the biological activity of the MPG1 hydrophobin during spore formation and plant infection.     

The mechanism and control of cytokinesis

Cytokinesis is under active investigation in each of the dominant experimental model systems. During 1996 and 1997, several developments necessitated the reassessment of the prevailing model for cytokinesis. In addition, the inventory of proteins required for cytokinesis has grown considerably. However, a molecular understanding of cytokinesis still remains elusive.     

Actin in penetration pegs of the fungal rice blast pathogen,Magnaporthe grisea

Summary The penetration peg is the structure used byMagnaporthe grisea to pierce the surface of rice leaves or very hard nonbiodegradable substrates. Penetration pegs produced by appressoria in vitro were examined by electron microscopy and immunofluorescence microscopy using various fluorophore labeled anti-actins. Freeze-substitution preparation of appressoria at early stages of substrate penetration showed that peg cytoplasm consisted primarily of a zone of exclusion, excluding even ribosomes, and was continuous with a similar region in the appressorium. Apical vesicles were, however, observed in short, presumably elongating pegs. Immunofluorescence microscopy was used to demonstrate binding of a monoclonal anti-actin to penetration peg cytoplasm, following permeabilization of appressoria by means of a brief sonication. Occasional filaments and ca. 300 nm diameter plaques were labeled in appressorial cytoplasm. Western blot analysis of germ tube extracts showed that the monoclonal probe bound predominantly to a single band with a molecular weight similar to that of rabbit muscle actin. Preincubation of the antibody with actin virtually eliminated peg labeling. We conclude that the penetration peg contains actin which may play a role in the formation of the zone of exclusion.Abbreviations PE polyethylene - Tris tris(hydroxymethyl)-amino-methane - TBS Tris-buffered saline - TBS-B Tris buffered saline plus 3% bovine serum albumin     

Structure and dynamics of green fluorescent protein

Many marine organisms are luminescent. The proteins that produce the light include a primary light producer (aequorin or luciferase) and often a secondary photoprotein that red shifts the light for better penetration in the ocean. Green fluorescent protein is one such secondary protein. It is remarkable in that it autocatalyzes the formation of its own fluorophore and thus can be expressed in variety of organisms in its fluorescent form. The recent determination of its 3D structure and other physical characterizations are revealing its molecular mechanism of action     

Physical mapping of the mouse tilted locus identifies an association between human deafness loci DFNA6/14 and vestibular system development

The tilted (tlt) mouse carries a recessive mutation causing vestibular dysfunction. The defect in tlt homozygous mice is limited to the utricle and saccule of the inner ear, which completely lack otoconia. Genetic mapping of tlt placed it in a region orthologous with human 4p16.3-p15 that contains two loci, DFNA6 and DFNA14, responsible for autosomal dominant, nonsyndromic hereditary hearing impairment. To identify a possible relationship between tlt in mice and DFNA6 and DFNA14 in humans, we have refined the mouse genetic map, assembled a BAC contig spanning the tlt locus, and developed a comprehensive comparative map between mouse and human. We have determined the position of tlt relative to 17 mouse chromosome 5 genes with orthologous loci in the human 4p16.3-p15 region. This analysis identified an inversion between the mouse and human genomes that places tlt and DFNA6/14 in close proximity.     

Insights into the mechanisms of homologous recombination from the structure of RuvA

The recent structure determination of RuvA has provided the first insights into the structural basis for its interaction with Holliday junction DNA. Multiple copies of a helix-hairpin-helix motif which line the four grooves between the monomers in the tetrameric structure are thought to be involved in the interaction of the protein with its DNA target. This suggests that the four arms of the junction are held by RuvA in a fourfold symmetric arrangement and has fuelled ideas on the way in which components of the Ruv complex combine to catalyse the process of homologous recombination