Molecular characterization of the proteinase-encoding gene, prb1, related to mycoparasitism by Trichoderma harzianum

The soil fungus Trichoderma harzianum is a mycoparasitic fungus known for its use as a biocontrol agent of phytopathogenic fungi. Among other factors, Trichoderma produces a series of antibiotics and fungal cell wall-degrading enzymes. These enzymes are believed to play an important role in mycoparasitism. Among the hydrolytic enzymes, we have identified a basic proteinase (Prb1) which is induced by either autoclaved mycelia, fungal cell wall preparation or chitin; however, the induction does not occur in the presence of glucose. The proteinase was purified and biochemically characterized as a serine proteinase of 31 kDa and pl 9.2. Based on the sequence of three internal peptides, synthetic oligonudeotide probes were designed. These probes allowed subsequent isolation of a cDNA and its corresponding genomic clone. The deduced amino acid sequence indicates that the proteinase is synthesized as a pre-proenzyme and allows its classification as a serine proteinase. Northen analysis shows that the induction of this enzyme is due to an increase in the corresponding mRNA level.     

Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host.

Infection by pathogenic fungi involves breaching the outer layer of the host by either mechanical or enzymatic means. Subtilisin-like proteinases are considered to be important in the infection process of entomopathogenic, nematophagous, and mycoparasitic fungi. Little is known regarding the expression of such proteinases by plant pathogenic fungi. Magnaporthe poae, a fungal pathogen of Kentucky bluegrass, expressed a subtilisin-like proteinase, proteinase Mp1, in the infected roots. Antibody was produced against the purified enzyme. From immunoblot analysis, expression of the proteinase in infected roots correlated with increasing severity of disease symptoms. Sequence analysis of a genomic clone indicated proteinase Mp1 was homologous to other fungal subtilisin-like proteinases. DNA gel blot analysis indicated proteinase Mp1 was encoded by a small gene family.     

Functional characterization of pathogen-responsive protein AtDabb1 with an antifungal activity from Arabidopsis thaliana

A plant antifungal protein was purified from Arabidopsis thaliana leaves by using a typical procedure consisting of anion exchange chromatography and high-performance liquid chromatography. We determined the amino acid sequence of the purified protein using MALDI-TOF/MS analysis, and found that the sequence matched that of a hypothetical Arabidopsis protein in GenBank (accession number NP_175547). We designated the protein as AtDabb1. After the cDNA encoding the AtDabb1 gene was cloned from an Arabidopsis leaf cDNA library, the recombinant protein was expressed in Escherichia coli and found to significantly inhibit cell growth of various pathogenic fungal strains. mRNA expression of the AtDabb1 gene was induced by pathogen-related signaling molecules including salicylic acid and jasmonic acid. These results suggest that AtDabb1 may contribute to the induced plant defense mechanism against diverse pathogenic fungi.     

An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR

To date, no single reported DNA extraction method is suitable for the efficient extraction of DNA from all fungal species. The efficiency of extraction is of particular importance in PCR-based medical diagnostic applications where the quantity of fungus in a tissue biopsy may be limited. We subjected 16 medically relevant fungi to physical, chemical and enzymatic cell wall disruption methods which constitutes the first step in extracting DNA. Examination by light microscopy showed that grinding with mortar and pestle was the most efficient means of disrupting the rigid fungal cell walls of hyphae and conidia. We then trialled several published DNA isolation protocols to ascertain the most efficient method of extraction. Optimal extraction was achieved by incorporating a lyticase and proteinase K enzymatic digestion step and adapting a DNA extraction procedure from a commercial kit (MO BIO) to generate high yields of high quality DNA from all 16 species. DNA quality was confirmed by the successful PCR amplification of the conserved region of the fungal 18S small-subunit rRNA multicopy gene.     

The human "peripheral-type" benzodiazepine receptor: regional mapping of the gene and characterization of the receptor expressed from cDNA.

A cDNA for the human "peripheral-type" benzodiazepine receptor (PBR) was isolated from a liver cDNA library. The 851-nucleotide probe hybridized with a approximately 1 kb mRNA in Northern blots of RNA extracted from various human tissues and cell lines. The human PBR probe was hybridized to DNA from a somatic cell hybrid mapping panel to determine that the gene maps to chromosome 22. With a regional mapping panel for chromosome 22, we localized the gene within band 22q13.31. The ligand-binding properties of the receptor expressed from the cDNA were examined in transient expression experiments and compared to the endogenous human PBR. The PBR ligand [3H]PK 11195 had high affinity for the expressed receptor in COS-1 cells, but the affinities of a pair of isoquinoline propanamide enantiomers differed remarkably in expressed and endogenous human PBR. These findings reveal that the host cell and/or post-translational modification may have an important influence on PBR function.     

cDNA cloning of mRNAs induced in resistant barley during infection by Erysiphe graminis f.sp. Hordei

Summary Near-isogenic cultivars of Hordeum vulgare which differ for the Mlp gene for resistance to Erysiphe graminis f.sp. hordei were inoculated with race 3 of this pathogen and in vitro translation products of mRNA populations compared by 2-dimensional gel electrophoresis and fluorography. This revealed the presence of new mRNA species in infected leaves compared to non-inoculated controls. These new mRNA species were more abundant in resistant leaves than susceptible leaves. A cDNA library was prepared from poly(A)⁺RNA isolated from infected leaves carrying the Mlp gene for resistance (cvMlp). The library was screened by differential hybridization using [³²P]-labelled cDNA prepared from poly(A)⁺RNA of both control and infected leaves. Six cDNA clones showing greater hybridization to cDNA prepared from infected leaves were selected. These six cDNA clones hybridized to DNA isolated from barley leaves but not to DNA from conidia of the fungus. In Northern blot analysis of RNA from infected leaves the six cDNA clones each hybridized to mRNA species of different size. Translation products for three of the cDNA clones corresponded to infection-related translation products identified on 2-dimensional fluorograms. The cDNA clones were used to study the kinetics of host mRNA induction during infection of the near-isogenic cultivars of barley. The host mRNA species corresponding to the cDNA clones were induced prior to 24 h after inoculation during the primary penetration processes. In addition the mRNAs corresponding to four of the cDNA clones increased to greater amounts in cvMlp than in the near-isogenic susceptible cultivar (cvmlp) over a 2-d period following inoculation. These results suggest that the Mlp gene has a regulatory role in host gene expression resulting in enhanced expression of several host mRNA species following infection by the powdery mildew fungus.     

Induction of thaumatin‐like proteins (TLPs) in Rhizoctonia solani‐infected rice and characterization of two new cDNA clones

Thaumatin‐like proteins (TLPs) were shown to be induced in rice plants (cv. IR58) that were infected with the sheath blight fungus, Rhizoctonia solani . Western blot analysis revealed the presence of two TLPs with sizes of 25 and 24 kDa which are different from a previously reported TLP with a size of 15.6 kDa from rice plants infiltrated with the non‐pathogenic bacterium, Pseudomonas syringae pv. syringae . By probing a cDNA expression library prepared from RNA isolated from R. solani ‐infected rice plants with a TLP antibody, several putative TLP cDNA clones were isolated and sequenced. The cDNA clones appeared to be derived from two different genes which shared only 77% sequence identity with each other and a lower percentage of sequence identity with the previously reported TLP cDNA clone. Southern blot analysis with the two TLP cDNAs revealed different rice genomic DNA fragments. Northern blot analysis also confirmed that a 1.1‐kb RNA detectable by the TLP cDNA inserts was induced by fungal infection. Thus rice TLPs are encoded by a family of at least three genes which are differentially expressed in responses to bacterial or fungal pathogens.     

低毒病毒及板栗疫病菌低毒力机制

低毒病毒是一类存在于板栗疫病菌细胞质中自主复制的无衣壳正链RNA病毒.感染低毒病毒后,板栗疫病菌的致病力显著降低,色素分泌减少,菌丝体由感染病毒前的桔黄色变为白色,产孢量降低或不产孢,漆酶表达水平明显下降.低毒病毒侵染性克隆的获得以及高效转化和转染体系的建立,使得低毒病毒成为目前唯一可以进行全面遗传操作的真菌病毒.利用低毒病毒作为探针来探测板栗疫病菌的致病力组成和毒力调节机制,已获得了一些很有意义的发现.本文介绍近几年低毒病毒及其与真菌相互作用的研究进展,包括低毒病毒的基因组和功能基因研究、低毒病毒和线粒体损害引起的板栗疫病菌低毒力机制、板栗疫病菌的RNA沉默系统以及低毒病毒抗RNA沉默的机制.低毒病毒/板栗疫病菌系统已经成为研究病毒与宿主相互作用以及病原真菌致病机理的很好的模式系统.     

MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization

Fungal hydrophobins are implicated in cell morphogenesis and pathogenicity in several plant pathogenic fungi including the rice blast fungus Magnaporthe grisea. A cDNA clone encoding a hydrophobin (magnaporin, MHP1) was isolated from a cDNA library constructed from rice leaves infected by M. grisea. The MHP1 codes for a typical fungal hydrophobin of 102 amino acids containing eight cysteine residues spaced in a conserved pattern. Hydropathy analysis of amino acids revealed that MHP1 belongs to the class II group of hydrophobins. The amino acid sequence of MHP1 exhibited about 20% similarity to MPG1, an M. grisea class I hydrophobin. Expression of MHP1 was highly induced during plant colonization and conidiation, but could hardly be detected during mycelial growth. Transformants in which MHP1 was inactivated by targeted gene replacement showed a detergent wettable phenotype, but were not altered in wettability with water. mhp1 mutants also exhibited pleiotropic effects on fungal morphogenesis, including reduction in conidiation, conidial germination, appressorium development and infectious growth in host cells. Furthermore, conidia of mhp1 mutants were defective in their cellular organelles and rapidly lose viability. As a result, mhp1 mutants exhibited a reduced ability to infect and colonize a susceptible rice cultivar. These phenotypes were recovered by re-introduction of an intact copy of MHP1. Taken together, these results indicate that MHP1 has essential roles in surface hydrophobicity and infection-related fungal development, and is required for pathogenicity of M. grisea.     

Expressed genes in the extraradical hyphae of an arbuscular mycorrhizal fungus, Glomus intraradices, in the symbiotic phase

To collect extraradical hyphae of arbuscular mycorrhizal (AM) fungi for RNA isolation, a PVDF membrane was laid on the hyphal compartment of a two-compartment culture system of transformed carrot hairy roots and Glomus intraradices. Extraradical hyphae free from host tissue were easily collected, and their RNA was rapidly extracted with a modified acid guanidinium thiocyanate-phenol-chloroform method. A 3'-RACE (rapid amplification of cDNA ends) of a known gene indicated that this protocol enabled the isolation of mRNA molecules as small as 2.3 kb. The cDNA libraries of an AM fungus from the aseptic extraradical hyphae in a symbiotic state were constructed for the first time. Three-fourth of 150 ESTs (expressed sequence tags) indicated low or no similarities to known sequences from other organisms.     

Phylogenetic congruence between subtropical trees and their associated fungi

Recent studies have detected phylogenetic signals in pathogen–host networks for both soil‐borne and leaf‐infecting fungi, suggesting that pathogenic fungi may track or coevolve with their preferred hosts. However, a phylogenetically concordant relationship between multiple hosts and multiple fungi in has rarely been investigated. Using next‐generation high‐throughput DNA sequencing techniques, we analyzed fungal taxa associated with diseased leaves, rotten seeds, and infected seedlings of subtropical trees. We compared the topologies of the phylogenetic trees of the soil and foliar fungi based on the internal transcribed spacer (ITS) region with the phylogeny of host tree species based on matK, rbcL, atpB, and 5.8S genes. We identified 37 foliar and 103 soil pathogenic fungi belonging to the Ascomycota and Basidiomycota phyla and detected significantly nonrandom host–fungus combinations, which clustered on both the fungus phylogeny and the host phylogeny. The explicit evidence of congruent phylogenies between tree hosts and their potential fungal pathogens suggests either diffuse coevolution among the plant–fungal interaction networks or that the distribution of fungal species tracked spatially associated hosts with phylogenetically conserved traits and habitat preferences. Phylogenetic conservatism in plant–fungal interactions within a local community promotes host and parasite specificity, which is integral to the important role of fungi in promoting species coexistence and maintaining biodiversity of forest communities.     

Antifungal activity of rice Pex5p, a receptor for peroxisomal matrix proteins

We have purified a novel antifungal protein from blast fungus (Magnaporthe grisea)-treated rice leaves using consecutive chromatographies on CM-Sepharose ion-change, Affi-gel blue, and HPLC gel filtration columns. We determined the N-terminal peptide sequence of the purified protein and subjected it to the NCBI/BLAST database and found the protein to be a partial fragment of the peroxisomal receptor protein in rice (OsPex5p). After cloning two cDNAs encoding OsPEX5L and OsPEX5S genes that are splice variants of OsPEX5 from a rice leaf cDNA library, we investigated their antifungal properties. The recombinant proteins were expressed in Escherichia coli and found to significantly inhibit cell growth of various pathogenic fungal strains. mRNA expression of the OsPEX5L gene was induced by diverse external stresses such as rice blast fungus, fungal elicitor, and other signaling molecules including H(2)O(2), abscisic acid, jasmonic acid, and salicylic acid. These results suggest that the peroxisomal receptor protein, OsPex5p, plays a critical role in the rice defense system against diverse external stresses including fungal pathogenic attack.     

微量RNA的cDNA PCR文库的构建

使用PCR（polymerase chain reaction)技术,调制了mRNA的cDNA PCR文库,实验证明,cDNA PCR文库能使原cDNA的量放大数百倍,同时,使用人体K562培养细胞的总RNA,对cDNA PCR文库法和反转录中的β-Actin的cDNA量进行了比较,cDNA PCR文库法中的β-Actin的cDNA量大于高于反转录中的β-Actin的cDNA量,使用75pg的人体K562培养细胞的总RNA,调制成50ul的CDNA PCR文库,使用1ul的CDNA PCR文库进行了PCR反应时,可对文库中β-Actin的CDNA进行PCR检测,因此,CDNA PCR文库显示了良好的信息放大性能。     

Generation of IL-23 producing dendritic cells (DCs) by airborne fungi regulates fungal pathogenicity via the induction of T(H)-17 responses

Interleukin-17 (IL-17) producing T helper cells (T(H)-17) comprise a newly recognized T cell subset with an emerging role in adaptive immunity to a variety of fungi. Whether different airborne fungi trigger a common signaling pathway for T(H)-17 induction, and whether this ability is related to the inherent pathogenic behavior of each fungus is currently unknown. Here we show that, as opposed to primary pathogenic fungi (Histoplasma capsulatum), opportunistic fungal pathogens (Aspergillus and Rhizopus) trigger a common innate sensing pathway in human dendritic cells (DCs) that results in robust production of IL-23 and drives T(H)-17 responses. This response requires activation of dectin-1 by the fungal cell wall polysaccharide b-glucan that is selectively exposed during the invasive growth of opportunistic fungi. Notably, unmasking of b-glucan in the cell wall of a mutant of Histoplasma not only abrogates the pathogenicity of this fungus, but also triggers the induction of IL-23 producing DCs. Thus, b-glucan exposure in the fungal cell wall is essential for the induction of IL-23/T(H)-17 axis and may represent a key factor that regulates protective immunity to opportunistic but not pathogenic fungi.     

A novel gene,CBP1, encoding a putative extracellular chitin-binding protein,may play an important role in the hydrophobic surface sensing of Magnaporthe grisea during appressorium differentiation

The conidial germ tube of the rice blast fungus, Magnaporthe grisea, differentiates a specialized cell, an appressorium, required for penetration into the host plant. Formation of the appressorium is also observed on artificial solid substrata such as polycarbonate. A novel emerging germ tube-specific gene, CBP1 (chitin-binding protein), was found in a cDNA subtractive differential library. CBP1 coded for a putative extracellular protein (signal peptide) with two similar chitin-binding domains at both ends of a central domain with homology to fungal chitin deacetylases and with a C-terminus domain rich in Ser/Thr related extracellular matrix protein such as agglutinin. The consensus sequence of the chitin-binding domain found in CBP1 has never been reported in fungi and is similar to the chitin-binding motif in plant lectins and plant chitinases classes I and IV. CBPI was disrupted in order to identify its function. Null mutants of CBP1 failed to differentiate appressoria normally on artificial surface but succeeded in normally differentiating appressoria on the plant leaf surface. Since the null mutant Cbp1- showed abnormal appressorium differentiation only on artificial surfaces and was sensitive to the chemical inducers, CBP1 seemed to play an important role in the recognition of physical factors on solid surfaces.