A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae * §

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1×106 ml−1, the percentages of conidium germination and appressorium formation were (97.98±0.67)% and (97.88±0.45)%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 μg total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA (complementary DNA) library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection

Application of cDNA array for studying the gene expression profile of mature appressoria of Magnaporthe grisea

Appressorium is an infection structure of the phytopathogenic fungus Magnaporthe grisea. Analysis of gene expression profiles of appressorium development provides insight into the molecular basis of pathogenicity and control of this fungal plant disease. A cDNA array representing 2927 unique genes based on a large EST (expressed sequence tag) database of M. grisea strain Y34 was constructed and used to profile the gene expression patterns at mycelium and appressorium maturation stages. Compared with mycelia, 55 up-regulated and 22 down-regulated genes were identified in mature appressoria. Among 77 genes, 16 genes showed no similarity to the genome sequences of M. grisea. A novel homologue of peptidyl-prolyl cis-trans isomerase was found to be expressed at low-level in mature appressoria of M. grisea. The results indicated that the genes such as pyruvate carboxylase, phospholipid metabolism-related protein and glyceraldehyde 3-phosphate dehydrogenase involved in gluconeogenesis, lipid metabolism and glycolysis, showed differential expression in mature appressoria. Furthermore, genes such as PTH11, beta subunit of G protein and SGT1 involved in cell signalling, were expressed differentially in mature appressoria. Northern blot analysis was used to confirm the cDNA array results

Reorientation of Mispositioned Spindles in Short Astral Microtubule Mutant spc72Δ Is Dependent on Spindle Pole Body Outer Plaque and Kar3 Motor Protein

Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules emanating from the spindle pole bodies (SPBs). Herein, we show by in vivo fluorescence microscopy that cells lacking Spc72, the SPB receptor of the cytoplasmic γ-tubulin complex, can only generate very short (<1 μm) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However, SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells, permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data exclude involvement of Spc72 in spindle formation and elongation functions

Cnm67p Is a Spacer Protein of the Saccharomyces cerevisiae Spindle Pole Body Outer Plaque

In Saccharomyces cerevisiae, the spindle pole body (SPB) is the functional homolog of the mammalian centrosome, responsible for the organization of the tubulin cytoskeleton. Cytoplasmic (astral) microtubules essential for the proper segregation of the nucleus into the daughter cell are attached at the outer plaque on the SPB cytoplasmic face. Previously, it has been shown that Cnm67p is an integral component of this structure; cells deleted for CNM67 are lacking the SPB outer plaque and thus experience severe nuclear migration defects. With the use of partial deletion mutants of CNM67, we show that the N- and C-terminal domains of the protein are important for nuclear migration. The C terminus, not the N terminus, is essential for Cnm67p localization to the SPB. On the other hand, only the N terminus is subject to protein phosphorylation of a yet unknown function. Electron microscopy of SPB serial thin sections reveals that deletion of the N- or C-terminal domains disturbs outer plaque formation, whereas mutations in the central coiled-coil domain of Cnm67p change the distance between the SPB core and the outer plaque. We conclude that Cnm67p is the protein that connects the outer plaque to the central plaque embedded in the nuclear envelope, adjusting the space between them by the length of its coiled-coil

Supercurrents in Magnesium Diboride/Metal Composite Wire

We have fabricated a series of ex situ copper sheathed powder-in-tube MgB2 wires with 20% by volume Ag, Pb, In, and Ga metal added to the MgB2 powder. We find the transport critical current of these wires increases significantly with the addition of specific metals to the core filament. In particular, the critical current density (Jc) of the MgB2/Ga(20%) wire is in excess of 5x10^4 A/cm^2 at 10K in self field, nearly 50 times that of the MgB2/Ag(20%) wire. The temperature dependent Jc of all wires is well described as an ensemble of clean S/N/S junctions in which the relevant parameters are the average thickness of the N layer, the critical temperature of the S layer, and a scaling term related to Jc at zero temperature. Eliminating the differences in the filament microstructure as the primary cause of the enhanced Jc, we suggest that Jc is determined by the magnitude of the proximity effect induced superconductivity in the normal metal layer, which is known to be proportional to the electron-electron interaction in N. We present one-dimensional material specific calculations that support this, and zero-field cooled DC magnetic susceptibility data that confirm an increased number of well-connected superconducting grains exist in the composite wires that contain metal additions with large electron-electron interactions and long electron mean free paths.Comment: 36 pages, 10 figures, 3 table

Predicting diffusivities in dense fluid mixtures

In this work the Enskog solution of the Boltzmann equation, as corrected by Speedy, together with the Weeks-Chandler-Andersen (WCA) perturbation theory of liquids is employed in correlating and predicting self-diffusivities of dense fluids. Afterwards this theory is used to estimate mutual diffusion coefficients of solutes at infinite dilution in sub and supercritical solvents. We have also investigated the behavior of Fick diffusion coefficients in the proximity of a binary vapor-liquid critical point since this subject is of great interest for extraction purposes. The approach presented here, which makes use of a density and temperature dependent hard-sphere diameter, is shown to be excellent for predicting diffusivities in dense pure fluids and fluid mixtures. The calculations involved highly nonideal mixtures as well as systems with high molecular asymmetry. The predicted diffusivities are in good agreement with the experimental data for the pure and binary systems. The methodology proposed here makes only use of pure component information and density of mixtures. The simple algebraic relations are proposed without any binary adjustable parameters and can be readily used for estimating diffusivities in multicomponent mixtures

Time-Lapse Video Microscopy Analysis Reveals Astral Microtubule Detachment in the Yeast Spindle Pole Mutant cnm67

Saccharomyces cerevisiae cnm67Δ cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein–labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the γ-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Δ cells Spc72–γ-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Δ cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs