Chemical constituents and cytotoxic activity from the wood-decaying fungus Xylaria sp. SWUF08-37.

A new cyclic pentapeptide, pentaminolarin (1), and a new cytochalasin, xylochalasin (2), along with thirteen known compounds (3-15) were isolated from the wood-decaying fungus Xylaria sp. SWUF08-37. The absolute configurations of 1 were determined by a combination of Marfey's method and TDDFT ECD calculation and the absolute configurations of 2 were established by TDDFT ECD calculation. Compound 12 showed moderate cytotoxicity against HeLa (IC50 = 19.60 µg/mL), HT29 (IC50 = 17.31 µg/mL), HCT116 (IC50 = 14.28 µg/mL), MCF-7 (IC50 = 15.38 µg/mL), and Vero (IC50 = 24.97 µg/mL) cell lines by MTT assay. Compounds 1 and 2 showed slight cytotoxicity against all tested cancer cell lines.

Differential Proteome Analysis of Hybrid Bamboo (Bambusa pervariabilis × Dendrocalamopsis grandis) Under Fungal Stress (Arthrinium phaeospermum).

In this study, TMT (tandem mass tag)-labeled quantitative protein technology combined with LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) was used to isolate and identify the proteins of the hybrid bamboo (Bambusa pervariabilis × Dendrocalamopsis grandis) and the bamboo inoculated with the pathogenic fungi Arthrinium phaeospermum. A total of 3320 unique peptide fragments were identified after inoculation with either A. phaeospermum or sterile water, and 1791 proteins were quantified. A total of 102 differentially expressed proteins were obtained, of which 66 differential proteins were upregulated and 36 downregulated in the treatment group. Annotation and enrichment analysis of these peptides and proteins using the GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases with bioinformatics software showed that the differentially expressed protein functional annotation items were mainly concentrated on biological processes and cell components. The LC-PRM/MS (liquid chromatography-parallel reaction monitoring/mass spectrometry) quantitative analysis technique was used to quantitatively analyze 11 differential candidate proteins obtained by TMT combined with LC-MS/MS. The up-down trend of 10 differential proteins in the PRM results was consistent with that of the TMT quantitative analysis. The coincidence rate of the two results was 91%, which confirmed the reliability of the proteomic results. Therefore, the differentially expressed proteins and signaling pathways discovered here may be the further concern for the bamboo-pathogen interaction studies.

Synchrotron X-ray micro-tomography imaging and analysis of wood degraded by Physisporinus vitreus and Xylaria longipes.

Incubation of Norway spruce with Physisporinus vitreus and sycamore with Xylaria longipes results in reduction in density of these wood species that are traditionally used for the top and bottom plate of a violin, which follows by enhanced acoustic properties. We used Synchrotron X-ray micro-tomography, to study the three-dimensional structure of wood at the micro-scale level and the alterations of the density distribution after incubation with two white-rot fungi. Micro-tomography data from wood treated at different incubation periods are analyzed and compared with untreated (control) specimens to determine the wood density map and changes at the cell-wall level. Differences between the density of early- and latewood, xylem ray and around bordered pits in both Norway spruce and sycamore are studied. Three-dimensional hyphal networks of the P.vitreus and Xylaria longipes hyphae are visualized inside the cell lumina and their significance on the density of the early- and latewood cells after different incubation periods are discussed. The study illustrates the utility of X-ray micro-tomography for both qualitative and quantitative studies of a wide variety of biological systems and due to its high sensitivity, small structural changes can be quantified.

Extensive alteration of fungal gene transcript accumulation and elevation of G-protein-regulated cAMP levels by a virulence-attenuating hypovirus.

Persistent infection of the chestnut blight fungus Cryphonectria parasitica with the prototypic hypovirus CHVI-713 results in attenuation of fungal virulence (hypo-virulence) and reduced accumulation of the GTP-binding (G) protein a subunit CPG-1. Transgenic cosuppression of CPG-1 accumulation in the absence of virus infection also confers hypovirulence. We now report the use of mRNA differential display to examine the extent to which virus infection alters fungal gene transcript accumulation and to assess the degree to which modification of CPG-1 signal transduction contributes to this alteration. More than 400 PCR products were identified that either increased (296 products) or decreased (127 products) in abundance as a result of virus infection. Significantly, 65% of these products exhibited similar changes as a result of CPG-1 cosuppression in the absence of virus infection. We also report that both virus infection and CPG-1 cosuppression elevate cAMP levels 3- to 5-fold. Additionally, it was possible to mimic the effect of virus infection and CPG-1 cosuppression on transcript accumulation for representative fungal genes by drug-induced elevation of cAMP levels. These results strengthen and extend previous indications that hypovirus infection causes a significant and persistent alteration of fungal gene expression/transcript accumulation. They further show that this alteration is primarily mediated through modification of the CPG-1 signaling pathway and suggest that, similar to mammalian Gi alpha subunits, CPG-1 functions as a negative modulator of adenylyl cyclase. Finally, these results suggest a role for G-protein-regulated cAMP accumulation in hypovirus-mediated alteration of fungal gene expression.

A viral dsRNA element of the chestnut blight fungus with a distinct genetic organization.

We have sequenced overlapping complementary DNA clones representing the viral double-stranded (ds) RNA from hypovirulent strain NB58 of the chestnut blight fungus Cryphonectria parasitica. Cryphonectria hypovirus 2-NB58 (CHV2-NB58) dsRNA contains 12,507 base pairs, excluding the poly(A) tail at the 3' end of the plus strand, and is organizationally similar to the largest dsRNA from the virus of strain EP713 (CHV1-713; identical to HAV; Shapira et al., (1991), EMBO J. 10, 731-739). CHV2-NB58 and CHV1-713 dsRNAs share approximately 60% nucleotide sequence identity. On the poly(A)-containing strand of CHV2-NB58, a 487-residue nontranslated region precedes two open reading frames, designated ORF A (438 codons) and ORF B (3291 codons). The connecting pentanucleotide sequence UAAUG (1802-1806) terminates ORF A and initiates ORF B. In contrast to the 69-kDa ORF A product of CHV1-713, the 50-kDa CHV2-NB58 ORF A product did not undergo autoproteolysis under the conditions tested, nor were motifs associated with cysteine proteases present in the CHV2-NB58 ORF A sequence. CHV2-NB58 ORF B products appear to be homologous with CHV1-713 ORF B products, and the motifs involved in autoproteolysis of the N-terminal 48 kDa of CHV1-713 ORF B were identified in the CHV2-NB58 ORF B product. Motifs associated with RNA polymerase and helicase activities were highly conserved between CHV2-NB58 and CHV1-713 and were found at similar genomic positions in the C-terminal half of ORF B.

Inhibition of ATPase activity in pea plasma membranes by fungal suppressors from Mycosphaerella pinodes and their peptide moieties.

The effects of two suppressors of the defense reactions of host plants, which had been purified from the pea pathogen Mycosphaerella pinodes, as well as the effects of peptide moieties, on the ATPase activity in pea plasma membranes were examined in vitro. One of the suppressors, Supprescin B, inhibited the ATPase activity in a non-competitive manner, but the other suppressor, Supprescin A, did not. Supprescin A was observed to reduce the inhibitory effect of Supprescin B. A tripeptide, Ser-Ser-Gly, and a hexapeptide, Ser-Ser-Gly-Asp-Glu-Thr, which were the respective peptide moieties of Supprescin A and B, inhibited the ATPase activity in a competitive manner. Supprescin B and fragments of the hexapeptide, such as Asp-Glu-Thr and Gly-Asp-Glu, inhibited not only the ATPase activity but also the acid phosphatase activity of plasma membranes in vitro. These results indicate that the acidic amino-acid residues of the "Asp-Glu" moiety seem to act as inhibitors of the phosphatase activity. Thus, the peptide moiety of Supprescin B consists of at least two functional elements.

Evidence for common ancestry of a chestnut blight hypovirulence-associated double-stranded RNA and a group of positive-strand RNA plant viruses.

Computer-assisted analysis of the putative polypeptide products encoded by the two open reading frames present in a large virus-like double-stranded RNA, L-dsRNA, associated with hypovirulence of the chestnut blight fungus, Cryphonectria parasitica, revealed five distinct domains with significant sequence similarity to previously described conserved domains within plant potyvirus-encoded polyproteins. These included the putative RNA-dependent RNA polymerase, RNA helicase, two papain-like cysteine proteases related to the potyvirus helper-component protease, and a cysteine-rich domain of unknown function similar to the N-terminal portion of the potyvirus helper-component protein. Phylogenetic trees derived from the alignment of the polymerase domains of L-dsRNA, a subset of positive-stranded RNA viruses, and double-stranded RNA viruses, using three independent algorithms, suggested that the hypovirulence-associated dsRNA and potyvirus genomes share a common ancestry. However, comparison of the organization of the conserved domains within the encoded polyproteins of the respective viruses indicated that the proposed subsequent evolution involved extensive genome rearrangement.

Two nonhomologus viruses of Cryphonectria (Endothia) parasitica reduce accumulation of specific virulence-associated polypeptides.

Double-stranded RNA responsible for transmissible hypovirulence of Cryphonectria (Endothia) parasitica affected the accumulation of specific polypeptides. Nonhomologous hypovirulence-causing double-stranded RNAs, originating in Europe or North America, affected accumulation of the same polypeptides. Fewer than 5% of detectable proteins were affected, indicating that hypovirulence is probably not the result of general debilitation of the fungus.