A prevention strategy against fungal attack for the conservation of cultural assets using a fungal index

The present study aimed to establish a prevention strategy to protect cultural assets from fungal attack. A fungal index that assesses conditions critical for fungal growth was determined using a fungal detector in the storerooms of historical buildings in Higashiomi area, Japan. The index measurements were repeated after 4 weeks' exposure of the detectors during the seasons when relative humidity outdoors and/or indoors was high. The index values obtained were from below the measurable lower limit to above the upper limit. The prevention strategy proposed was as follows. Each microclimate was categorized into three levels, A, B, or C, depending on the index values, <1.8, 1.8–18 or >18, respectively. If all microclimates in a room maintain level A continuously, the room is considered free of contamination. If some microclimates maintain level B, fungal contamination might occur. If microclimates maintain level C, fungal contamination is unavoidable, and countermeasures should be taken promptly. Finally, fungal indices are measured for evaluation of the countermeasures and for level-A confirmation. The systematic use of fungal indices will provide practically useful information for conservation and must be applicable to IPM (Integrated Pest Management) in museums and libraries.     

L3/Lhx8 is a pivotal factor for cholinergic differentiation of murine embryonic stem cells

L3/Lhx8 is a member of the LIM-homeobox gene family. Previously, we demonstrated that L3/Lhx8-null mice specifically lacked cholinergic neurons in the basal forebrain. In the present study, we conditionally suppressed L3/Lhx8 function during retinoic acid-induced neural differentiation of a murine embryonic stem (ES) cell line using an L3/Lhx8-targeted small interfering RNA (siRNA) produced by an H1.2 promoter-driven vector. Our culture conditions induced efficient differentiation of the ES cells into neurons and astrocytes, but far less efficient differentiation into oligodendrocytes. Suppression of L3/Lhx8 expression by siRNA led to a dramatic decrease in the number of cells positive for the cholinergic marker ChAT, and overexpression of L3/Lhx8 recovered this effect. However, no significant changes were observed in the number of Tuj1+ neurons and GABA+ cells. These results strongly suggest that L3/Lhx8 is a key factor in the cholinergic differentiation of murine ES cells and is involved in basal forebrain development.     

Competence of roots for race-specific resistance and the induction of acquired resistance against Magnaporthe oryzae

Generally, Magnaporthe oryzae , the causal agent of rice blast disease, is considered to be a typical leaf-infecting plant pathogenic fungus. However, it was recently reported that M. oryzae shares many characteristics in common with root-infecting pathogens and indeed was able to infect roots. Here, we report on studies testing for the capacity of roots of rice and barley to resist infections with M. oryzae . We established that roots of rice plants were colonized by M. oryzae in a manner which is different from the gene-for-gene specificity seen in leaves for the same genotypes. Furthermore, treatment of rice seedlings with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a chemical that protects leaves effectively against blast by conditioning acquired resistance, was not able to prevent colonization of roots by M. oryzae although a reduction in disease levels was observed. Moreover, BTH was not able to protect barley roots against infection with M. oryzae . Taken together, our results suggest that although roots show intrinsic variation in their ability to resist colonization by M. oryzae , neither gene-for-gene incompatibility nor aquired resistance are as effective at blocking the pathogen as they are in leaves.     

The tetraspanin gene ClPLS1 is essential for appressorium-mediated penetration of the fungal pathogen Colletotrichum lindemuthianum

Conservation of the molecular mechanisms controlling appressorium-mediated penetration during evolution was assessed through a functional study of the ClPLS1 gene from Colletotrichum lindemuthianum orthologous to the MgPLS1 from Magnaporthe grisea, involved in penetration peg development. These two plant-pathogenic Pyrenomycetes differentiate appressoria to penetrate into plant tissues. We showed that ClPLS1 is a functional homologue of MgPLS1 in M. grisea. Loss of ClPLS1 function had no effect on vegetative growth, conidiation or on appressorium differentiation and maturation. However, Clpls1::hph mutants are non-pathogenic on either intact or wounded bean leaves, as a result of a defect in the formation and/or positioning of the penetration pore and consequently in the formation of the penetration peg. These observations suggest that the fungal tetraspanins control a conserved appressorial function that could be required for the correct localization of the site where the penetration peg emerges.     

The outer membrane of Pseudomonas aeruginosa is a barrier against the penetration of disaccharides

The outer membrane of Pseudomonas aeruginosa acted as a barrier against the penetration of di- (Mr, 342), tri- (Mr, 504) and tetrasaccharides (Mr, 666), whereas the membrane allowed the penetration of pentose (Mr, 150) and methylhexoses (Mr, 194) into the periplasm. When the intact cells of P. aeruginosa were treated with 600 mosM saccharides of various sizes and observed under an electron microscope, saccharides of Mr larger than 342 caused the extensive shrinking of the outer membrane. Whereas the cells treated with the saccharides of Mr less than 194 or with sucrose in the presence of EDTA showed plasmolysis. Determination of the extent of saccharide penetration into the periplasm of the cells treated with 600 mosM sodium chloride or with 600 mosM saccharides of various sizes showed that only pentose and hexoses, so far examined, were penetrable but di-, tri- and tetrasaccharides were impenetrable.     

Plasma membrane-cell wall adhesion is required for expression of plant defense responses during fungal penetration

Fungal pathogens almost invariably trigger cell wall-associated defense responses, such as extracellular hydrogen peroxide generation and callose deposition, when they attempt to penetrate either resistant or susceptible plant cells. In the current study, we provide evidence that the expression of these defenses is dependent on adhesion between the plant cell wall and the plasma membrane. Peptides containing an Arg-Gly-Asp (RGD) motif, which interfered with plasma membrane-cell wall adhesion as shown by the loss of the thin plasma membrane-cell wall connections known as Hechtian strands, reduced the expression of cell wall-associated defense responses during the penetration of nonhost plants by biotrophic fungal pathogens. This reduction was associated with increased fungal penetration efficiency. Neither of these effects was seen after treatment with similar peptides lacking the RGD motif. Disruption of plant microfilaments had no effect on Hechtian strands but mimicked the effect of RGD peptides on wall defenses, suggesting that the expression of cell wall-associated defenses involves communication between the plant cell wall and the cytosol across the plasma membrane. To visualize the state of the plasma membrane-cell wall interaction during fungal penetration, we observed living cells during sucrose-induced plasmolysis. In interactions that were characterized by the early expression of cell wall-associated defenses, there was no change, or an increase, in plasma membrane-cell wall adhesion under the penetration point as the fungus grew through the plant cell wall. In contrast, for rust fungus interactions with host plants, there was a strong correlation between a lack of cell wall-associated defenses and a localized decrease in plasma membrane-cell wall adhesion under the penetration point. Abolition of this localized decreased adhesion by previous inoculation with a fungus that increased plasma membrane-cell wall adhesion resulted in reduced penetration by the rust fungus and induction of cell wall-associated defenses. These results suggest that rust fungi may induce a decrease in plasma membrane-cell wall adhesion as a means of disrupting the expression of nonspecific defense responses during penetration of host cells.     

Arginine 469 is a pivotal residue for the Hsc70-GlcNAc-binding property

The members of the 70 kDa-heat shock proteins (HSP70) family play numerous fundamental functions in the cell such as promoting the assembly of multimeric complexes or helping the correct folding of nascent proteins to take place. In numerous previous studies we demonstrated that Hsp70 and its constitutive isoform Hsc70 are endowed of a GlcNAc-binding activity. The molecular modeling of the substrate binding domain of Hsc70 and in silico docking experiments using Ser/Thr-O-GlcNAc motifs allowed to define the potential carbohydrate-recognition region and to point out the crucial position of Arg469 as an amino-acid directly interacting with the sugar moiety. We cloned a flagged Hsc70 in a pCMV.SPORT6 vector and we showed that the mutation R469A decreased the GlcNAc-binding property of the chaperone of around 70%. This is the first work reporting the localization of the GlcNAc-binding domain of a member of the HSP70 family.     

The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae

Attack by the host powdery mildew Erysiphe cichoracearum usually results in successful penetration and rapid proliferation of the fungus on Arabidopsis. By contrast, the nonhost barley powdery mildew Blumeria graminis f. sp. hordei (Bgh) typically fails to penetrate Arabidopsis epidermal cells. In both instances the plant secretes cell wall appositions or papillae beneath the penetration peg of the fungus. Genetic screens for mutations that result in increased penetration of Bgh on Arabidopsis have recently identified the PEN1 syntaxin. Here we examine the role of PEN1 and of its closest homologue, SYP122, identified as a syntaxin whose expression is responsive to infection. pen1 syp122 double mutants are both dwarfed and necrotic, suggesting that the two syntaxins have overlapping functions. Although syp122-1 and the cell wall mur mutants have considerably more pronounced primary cell wall defects than pen1 mutants, these have relatively subtle or no effects on penetration resistance. Upon fungal attack, PEN1 appears to be actively recruited to papillae, and there is a 2-h delay in papillae formation in the pen1-1 mutant. We conclude that SYP122 may have a general function in secretion, including a role in cell wall deposition. By contrast, PEN1 appears to have a basal function in secretion and a specialized defense-related function, being required for the polarized secretion events that give rise to papilla formation.     

Epstein-Barr virus induction by a serum factor. Purification of a high molecular weight protein that is responsible for induction

Serum contains a factor that induces Epstein-Barr virus antigens in latently infected human lymphoblastoid cell lines and that cooperates with chemical inducers. Here we report the purification of a protein that is responsible for the effect. Using ammonium sulphate precipitation, chromatography on DEAE-agarose and cellulose, molecular sieve chromatography on Bio-Gel A-5, and glycerol gradient centrifugation, the factor was enriched about 300,000-fold compared to calf serum. Under neutral conditions, the protein chromatographed as a high molecular weight protein of about 5.5 X 10(5) both in its active and inactive form. Both forms of the molecule sedimented at about 7 s. The factor is an acidic protein with a pI of 5. It seems to be composed of high molecular and low molecular weight subunits. Nanogram amounts of purified factor are sufficient for measurable inducing effects.     

Loss of epidermal integrity by T cell-mediated attack induces long term local resistance to subsequent attack. II. Thymus dependency in the induction of the resistance

Our previous study showed that in cutaneous graft-vs-host disease (GVHD) induced by intradermal injection of autoreactive T cells the epidermal structures spontaneously recovered from the destruction became resistant to the subsequent attempts to induce the cutaneous GVHD and that the resistance correlated well with a 30-fold increase in the number of Thy-1+ epidermal cells (Thy-1+EC). We show that the resistance to the cutaneous GVHD was never induced in athymic nude mice and adult thymectomy lethal radiation and bone marrow reconstitution (ATXBM) mice under the same conditions, indicating that the induction of the resistance is dependent on the presence of thymus. A great increase in the number of Thy-1+EC was similarly observed in the epidermis of the athymic nude and ATXBM mice that spontaneously recovered from the cutaneous GVHD and that remained susceptible to the induction of the cutaneous GVHD. However, the results with B10Thy-1.1----B6 radiation chimeras clearly demonstrate that the vast majority of the increased Thy-1+EC found in the "susceptible" epidermis of the ATXBM mice were of donor bone marrow origin and there was no increase in the number of host-derived Thy-1+EC, whereas in the "resistant" epidermis of the XBM mice both Thy-1+EC populations were equally increased. The overall results indicate that the expansion of Thy-1+EC that mature in the thymus is crucial to the induction of the resistance, although the migration of bone marrow-derived Thy-1+EC precursors to the epidermis occurs quite independently of the presence of thymus.     

A peroxiredoxin Q homolog from gentians is involved in both resistance against fungal disease and oxidative stress

An antifungal protein (GtAFP1) showing antimicrobial activity against phytopathogenic fungi was purified from leaves of Gentiana triflora. The deduced amino acid sequence of the cDNA of the corresponding gene, GtAFP1, showed 94, 75, 72 and 63% amino acid identities with peroxiredoxin Q from Populus balsamifera x P. deltoides subsp. trichocarpa, Sedum lineare, Suaeda maritima and Arabidopsis thaliana, respectively. The GtAFP1 gene is suggested to be present in the genome in one to two copies and was expressed in the leaves, roots and stems. Expression of GtAFP1 was induced by treatment with salicylic acid, but not methyl jasmonate. Recombinant GtAFP1 protein showed not only antifungal activity but also thioredoxin-dependent peroxidase activity. Overexpression of GtAFP1 in tobacco plants improved tolerance not only against fungal diseases but also against oxidative stress. These results indicate that GtAFP1 might act as a disease and oxidative stress defensive gene in plants and could be useful for engineering stress-resistant plants.     

A major function of the tobacco floral nectary is defense against microbial attack

 We have characterized the major nectar protein (Nectarin I) from ornamental tobacco as a superoxide dismutase that functions to generate high levels of hydrogen peroxide in nectar. Other nectar functions include an anti-polygalacturonase activity that may be due to a polygalacturonase inhibiting protein (PGIP). We also examined the expression of defense related genes in the nectary gland by two independent methods. We isolated a sample of nectary-expressed cDNAs and found that 21% of these cDNAs were defense related clones. Finally, we examined the expression of a number of specific defense-related genes by hybridization to specific cDNAs. These results demonstrated that a number of specific defense genes were more strongly expressed in the floral nectary than in the foliage. Taken together these results indicate that the floral nectary gland can have specific functions in plant defense. Received August 8, 2002; accepted January 7, 2003 Published online: June 2, 2003     

Dopamine is a key factor for the induction of egg diapause of the silkworm, Bombyx mori.

The silkworm, Bombyx mori, enters diapause in the early embryonic stage. Embryonic diapause is induced by incubating eggs of the maternal generation at high temperature (diapause type), whereas incubation at low temperature results in non-diapausing progeny (non-diapause type). Measurement of catecholamine concentrations in haemolymph and brain-subesophageal ganglia (Br-SGs) showed that only dopamine concentrations in both tissues are consistently higher in diapause-type than non-diapause-type larvae and pupae. In particular, the difference in dopamine concentrations in both tissues increases around pupal ecdysis. During the early pupal stage, Dopa decarboxylase activities and mRNA concentrations in Br-SGs were also much higher in diapause-type than non-diapause-type insects. Elevation of dopamine levels induced by feeding Dopa to penultimate-instar and last-instar larvae, and by injecting Dopa or dopamine into pupae 2 days after pupation made the non-diapause-destined insects lay diapause-destined eggs at 59% and approximately 70% frequencies, respectively. Furthermore, injection of Dopa or dopamine elevated mRNA levels of the diapause hormone in the Br-SGs of non-diapause-type pupae 1 day after injection. Incubation of Br-SGs isolated from non-diapause-type day-2 pupae with Dopa or dopamine also stimulated the expression of diapause hormone mRNA. These data indicate that environmental stimuli during embryonic development increase dopamine levels in both hemolymph and Br-SGs from the larval stage to early pupal stage, which results in laying of diapause-destined eggs by female adults through enhanced expression of the diapause hormone gene.