The state of proteome profiling in the fungal genus Aspergillus.

Aspergilli are an important genus of filamentous fungi that contribute to a multibillion dollar industry. Since many fungal genome sequencing were recently completed, it would be advantageous to profile their proteome to better understand the fungal cell factory. Here, we review proteomic data generated for the Aspergilli in recent years. Thus far, a combined total of 28 cell surface, 102 secreted and 139 intracellular proteins have been identified based on 10 different studies on Aspergillus proteomics. A summary proteome map highlighting identified proteins in major metabolic pathway is presented.     

VeA调控曲霉发育致病的研究进展

全局性调控因子Ve A仅在真菌中存在,具有保守性。Ve A参与细胞的多过程调控,包括发育分化、次生代谢、氧化胁迫应答、侵染宿主致毒致病等。文中总结曲霉Ve A的相关调控功能和作用机理研究,以利于防控真菌传播存活及致毒致病措施的设计,促进抗真菌作物的育种,减少作物被曲霉等真菌及毒素污染;还提出进一步开展ve A基因调控功能研究的方向和作为防控真菌污染靶位点的应用策略。     

Conservative production of galactosaminogalactan in Metarhizium is responsible for appressorium mucilage production and topical infection of insect hosts

The exopolysaccharide galactosaminogalactan (GAG) has been well characterized in Aspergilli, especially the human pathogen Aspergillus fumigatus. It has been found that a five-gene cluster is responsible for GAG biosynthesis in Aspergilli to mediate fungal adherence, biofilm formation, immunosuppression or induction of host immune defences. Herein, we report the presence of the conserved GAG biosynthetic gene cluster in the insect pathogenic fungus Metarhizium robertsii to mediate either similar or unique biological functions. Deletion of the gene cluster disabled fungal ability to produce GAG on germ tubes, mycelia and appressoria. Relative to the wild type strain, null mutant was impaired in topical infection but not injection of insect hosts. We found that GAG production by Metarhizium is partially acetylated and could mediate fungal adherence to hydrophobic insect cuticles, biofilm formation, and penetration of insect cuticles. In particular, it was first confirmed that this exopolymer is responsible for the formation of appressorium mucilage, the essential extracellular matrix formed along with the infection structure differentiation to mediate cell attachment and expression of cuticle degrading enzymes. In contrast to its production during A. fumigatus invasive growth, GAG is not produced on the Metarhizium cells harvested from insect hemocoels; however, the polymer can glue germ tubes into aggregates to form mycelium pellets in liquid culture. The results of this study unravel the biosynthesis and unique function of GAG in a fungal system apart from the aspergilli species.     

Studies on aspergilli XVII. Effect of nitrogen sources on growth and fruiting

Summary The effect of ammonium, nitrate, and organic nitrogen on growth and sporulation of 18 Aspergilli was examined in a chemically defined medium in surface culture under controlled conditions. All three forms of nitrogen were metabolized by all the Aspergilli tested. Ammonium nitrogen was not good both for growth and fruiting. This was due to the sharp fall in the pH level which resulted due to the rapid utilization of anions of the ammonium nitrogen than cations. The effect of adding succinic acid in the medium containing ammonium nitrogen has been discussed.Good growth of Aspergilli in media containing nitrate nitrogen with the accompanying rise in the pH of the medium showed that these species are capable of reducing nitrate nitrogen to the level of ammonia. The role of succinic acid in the utilization of nitrate nitrogen was investigated. All fungi accomplished good growth on a medium containing asparagine.     

真菌遗传转化系统的研究进展

真菌遗传转化是功能基因研究的重要方法,就真菌遗传转化系统的最新研究进展进行了综述,主要包括真菌遗传转化的方法、各种方法的优缺点、选择标记的种类、标记基因的分离方法以及转化系统的应用等。     

The VeA regulatory system and its role in morphological and chemical development in fungi

In fungi, the velvet gene, or veA, is involved in the regulation of diverse cellular processes, including control of asexual and sexual development as well as secondary metabolism. This global regulator is conserved in numerous fungal species. Interestingly, in Aspergilli, where most of the studies on veA have been carried out, this gene has been described to mediate development in response to light. In recent years the knowledge of this important regulatory system has expanded through the use of Aspergillus nidulans as a model organism, and through the study of veA orthologs across fungal genera. This review includes information on the current understanding of veA function and its mechanism of action. The fact that veA has only been found in fungi, together with advances in the elucidation of the veA mechanism, might be useful in designing future control strategies to decrease the detrimental effects of fungi while enhancing those qualities that are beneficial.     

Prevalence of airborne Aspergillus flavus in Khartoum (Sudan) Airspora with reference to dusty weather and inoculum survival in simulated summer conditions

Khartoum air was scanned for airborne Aspergillus flavus for 12 months using the horizontal gravitational settling method. Frequency of occurrence was related to total fungal catch and dusty weather. The Aspergilli were prevalent (68% of total isolated/plate/month) and A. flavus constituted 31% of the total Aspergilli. In June (hot, dry & dusty) Aspergilli constituted 79% of the total isolates, whilst A. flavus represented 30% from amongst the other Aspergilli. A. flavus, A. niger, A. nidulans (conidial & ascosporic states), A. terreus, Eurotium amstelodami and A. fumigatus, in descending order of prevalence were isolated in June. Other pathogenic or potentially pathogenic forms, isolated, were Cladosporium, Curvularia and Penicillium. Amongst winter isolations A. flavus was sporadic to absent in occurrence. A. flavus spore inocula that underwent hourly intermitted exposure to 45 °C, showed a decrease in spore germinability as well as reduced germ length.     

Diverse and bioactive endophytic Aspergilli inhabit Cupressaceae plant family

Aspergilli are filamentous, cosmopolitan and ubiquitous fungi which have significant impact on human, animal and plant welfare worldwide. Due to their extraordinary metabolic diversity, Aspergillus species are used in biotechnology for the production of a vast array of biomolecules. However, little is known about Aspergillus species that are able to adapt an endophytic lifestyle in Cupressaceae plant family and are capable of producing cytotoxic, antifungal and antibacterial metabolites. In this work, we report a possible ecological niche for pathogenic fungi such as Aspergillus fumigatus and Aspergillus flavus. Indeed, our findings indicate that A. fumigatus, A. flavus, Aspergillus niger var. niger and A. niger var. awamori adapt an endophytic lifestyle inside the Cupressaceous plants including Cupressus arizonica, Cupressus sempervirens var. fastigiata, Cupressus semipervirens var. cereiformis, and Thuja orientalis. In addition, we found that extracts of endophytic Aspergilli showed significant growth inhibition and cytotoxicity against the model fungus Pyricularia oryzae and bacteria such as Bacillus sp., Erwinia amylovora and Pseudomonas syringae. These endophytic Aspergilli also showed in vitro antifungal effects on the cypress fungal phytopathogens including Diplodia seriata, Phaeobotryon cupressi and Spencermartinsia viticola. In conclusion, our findings clearly support the endophytic association of Aspergilli with Cupressaceae plants and their possible role in protection of host plants against biotic stresses. Observed bioactivities of such endophytic Aspergilli may represent a significant potential for bioindustry and biocontrol applications.     

Studies on growth inhibition by lectins of penicillia and aspergilli

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [³H]acetate, N-acetyl-D-[³H]glucosamine and D-[¹⁴C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.Abbreviations Con A concanavalin A - PNA peanut agglutinin - SBA soybean agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine     

A new dominant selection marker for transformation of Pichia pastoris to soraphen A resistance

Despite the considerable progress molecular genetics of filamentous fungi has made during the past decade, there is still an urgent need for efficiently working selectable markers for fungal transformation. Using Pichia pastoris as a host, we describe the development of a new dominant selectable marker of prokaryotic origin. This system, termed sor(R), is based upon the resistance of the bacterial enzyme acetyl-CoA carboxylase (ACCase) to the macrocyclic polyketide soraphen A, a potent inhibitor of fungal ACCase produced by the myxobacterium Sorangium cellulosum. In this study, we firstly demonstrate that the integration of a single sor(R) cassette into the P. pastoris genome confers resistance to elevated concentrations of soraphen A. Furthermore, it has been shown that the versatility of this marker can be considerably increased by splitting the sor(R) cassette, especially when successive transformations are performed on the same strain. As pronounced sensitivity to soraphen A is the rule among filamentous fungi, we expect the sor(R) marker to be a widely applicable tool for fungal transformation.     

Development of ToxA and ToxB promoter-driven fluorescent protein expression vectors for use in filamentous ascomycetes

The green fluorescent protein (GFP) has been established as the premier in vivo reporter for investigations of gene expression, protein localization, and cell and organism dynamics. The fungal transformation vector pCT74, with sGFP under the control of the ToxA promoter from Pyrenophora tritici-repentis, effectively expresses GFP in a diverse group of filamentous ascomycetes. Due to the versatility of ToxA promoter-driven expression of GFP, we constructed an additional set of fluorescent protein expression vectors to expand the color palette of fluorescent markers for use in filamentous fungi. EYFP, ECFP and mRFP1 were successfully expressed from the ToxA promoter in its fungus of origin, P. tritici-repentis, and a distant relative, Verticillium dahliae. Additionally the ToxB promoter from P. tritici-repentis drove expression of sGFP in V. dahliae, suggesting a similar potential to the ToxA promoter for heterologous expression in ascomycetes. The suite of fungal transformation vectors presented here promise to be useful for a variety of fungal research applications.     

Early diagnosis of invasive fungal infections caused by Aspergillus and other emerging mycelial fungi

The usefulness of surrogate markers in the diagnosis of invasive fungal infections caused by Aspergillus and other emerging mycelial fungi is based on the ability of surrogate markers to detect the infection caused by different species of mycelial fungi. Conventional microbiological methods for diagnosis of fungal disease are slow and insensitive. Antigen based assays or measurement of (1-3)-beta-D-glucan in blood have been developed and validated in clinical laboratories. We review these diagnostic contemporary tools, their clinical application and impact.     

Species of Aspergillus causing post-harvest fruit decay in Israel

Five species ofAspergillus were isolated from post-harvest decays of pears, apples, peaches, grapes, strawberries, melons and tomatoes.A. niger was the most common species and was isolated from all types of fruits. Next in frequency wasA. flavus, which was infrequently isolated from stored grapes, tomatoes and peaches.A. wentii, A. ochraceus andA. tamarii were each cultured from only one type of stored fruit.All the fruit-decay Aspergilli were typical inhabitants of the atmospheres of store-rooms. Of these,A. niger was by far the most ubiquitous species, comprising about 80% of the total airborne Aspergilli. Each of the four other pathogenic Aspergilli comprised 0.2–0.4% of the total. However, some of the most common airborne Aspergilli such asA. versicolor (6.6% of the total), have never been isolated from any fruit decay and were also very limited in their potential pathogenicity.The highest potential pathogenicity was attributed toA. niger, which was capable of infecting all inoculated fruits except eggplants, followed by a 2–3-day incubation period. The other Aspergilli cultured, pathogenic as well as airborne, showed selective potential pathogenicity when artificially inoculated into the fruit. Fruit tissues of apples, pears and tomatoes, followed by those of peaches and grapes, were found the most suitable for Aspergilli development, whereas strawberries and, more so, eggplants were mostly resistant to infection.     

Challenges of microsatellite isolation in fungi

Although they represent powerful genetic markers in many fields of biology, microsatellites have been isolated in few fungal species. The aim of this study was to assess whether obtaining microsatellite markers with an acceptable level of polymorphism is generally harder from fungi than in other organisms. We therefore surveyed the number, nature and polymorphism level of published microsatellite markers in fungi from the literature and from our own data on seventeen fungal microsatellite-enriched libraries, and in five other phylogroups (angiosperms, insects, fishes, birds and mammals). Fungal microsatellites indeed appeared both harder to isolate and to exhibit lower polymorphism than in other organisms. This appeared to be due, at least in part, to genomic specificities, such as scarcity and shortness of fungal microsatellite loci. A correlation was observed between mean repeat number and mean allele number in the published fungal microsatellite loci. The cross-species transferability of fungal microsatellites also appeared lower than in other phylogroups. However, microsatellites have been useful in some fungal species. Thus, the considerable advantages of these markers make their development worthwhile, and this study provides some guidelines for their isolation.     

Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

Background  

The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene.     

Finding single copy genes out of sequenced genomes for multilocus phylogenetics in non-model fungi

Historically, fungal multigene phylogenies have been reconstructed based on a small number of commonly used genes. The availability of complete fungal genomes has given rise to a new wave of model organisms that provide large number of genes potentially useful for building robust gene genealogies. Unfortunately, cross-utilization of these resources to study phylogenetic relationships in the vast majority of non-model fungi (i.e. "orphan" species) remains an unexamined question. To address this problem, we developed a method coupled with a program named "PHYLORPH" (PHYLogenetic markers for ORPHans). The method screens fungal genomic databases (107 fungal genomes fully sequenced) for single copy genes that might be easily transferable and well suited for studies at low taxonomic levels (for example, in species complexes) in non-model fungal species. To maximize the chance to target genes with informative regions, PHYLORPH displays a graphical evaluation system based on the estimation of nucleotide divergence relative to substitution type. The usefulness of this approach was tested by developing markers in four non-model groups of fungal pathogens. For each pathogen considered, 7 to 40% of the 10-15 best candidate genes proposed by PHYLORPH yielded sequencing success. Levels of polymorphism of these genes were compared with those obtained for some genes traditionally used to build fungal phylogenies (e.g. nuclear rDNA, β-tubulin, γ-actin, Elongation factor EF-1α). These genes were ranked among the best-performing ones and resolved accurately taxa relationships in each of the four non-model groups of fungi considered. We envision that PHYLORPH will constitute a useful tool for obtaining new and accurate phylogenetic markers to resolve relationships between closely related non-model fungal species.     

Formation of arthroconidia during regeneration and selection of transformed Epichloë festucae protoplasts

Transformation is an essential tool for modern fungal research and has played a fundamental role in gaining insight into gene function. Polyethylene glycol (PEG)-mediated transformation of protoplasts is the most commonly used method for genetic transformation of filamentous fungi. Selectable marker genes, that confer resistance to antibiotics, are generally incorporated with the DNA of interest, allowing transformed cells to grow through the antibiotic overlay. Colonies arising from transformed fungal cells are sub-cultured and further analysed. However, the morphological state of the fungal cells during the transformation procedure has been largely overlooked. We investigated the morphological appearance of transformed fungal cells prior to their emergence through the antibiotic overlay. Hyphae appeared to segment and bulge, reminiscent of arthroconidia, an asexual spore typically produced by segmentation of pre-existing hyphae. Selective expression of eGFP under the control of a spore specific promoter, PcatA, in these cells confirmed their spore-like nature. Reducing the oxygen availability to surface-grown cultures partially recapitulated this morphological form. A GFP fusion to the cell wall integrity MAP kinase MpkA localised to the arthroconidia nuclei suggesting the cell wall integrity signalling pathway modulates cell wall stress responses in arthroconidia. This dramatic morphological change was also observed in transformed Magnaporthe oryzae cells suggesting it may be a more general phenomenon in filamentous fungi. Given the changes in cellular structure and spore-like appearance, these observations may have technical implications for deleting genes involved in these processes in Epichloë festucae and, more broadly, a range of fungal species.     

基因工程改良在昆虫病原真菌中的应用

昆虫病原真菌是自然界控制害虫种群的主要生物因子,其研究开发越来越受到重视。但由于存在致死速度慢、防效不稳定、对环境条件要求高等缺点,限制了昆虫病原真菌的应用。近年来,通过基因工程技术对昆虫病原真菌进行遗传改造,提高菌株的致病性和毒力,创造高效、稳定的工程菌株取得了较大进展。对昆虫病原真菌选择标记、遗传转化方法、基因工程改良及应用等方面最新研究进展进行了综述。     

Integration Efficiency in DNA-Induced Transformation of Pneumococcus. I. a Method of Transformation in Solid Medium and Its Use for Isolation of Transformation-Deficient and Recombination-Modified Mutants

A method of transformation on solid medium especially adapted for pneumococcus has been developed. Under specific conditions, all colonies that are allowed to grow in the presence of transforming DNA for six hours give rise to transformed bacteria. Combined with replica plating this technique has been used to isolate mutants modified with regard to recombination. Most of the mutants found are transformation-defective and show a large diversity in their response to ultraviolet light. Some of these mutants have lost their ability to take up transforming DNA. One shows a reduced yield of transformants for a given quantity of DNA taken up. Mutants that manifest altered behavior with regard to marker efficiencies have also been isolated. One of these exhibits a decrease in the transformation efficiency of only the high efficiency markers and two mutants show a decrease in the transformation efficiency of the low efficiency markers.