Dissecting the fungal biology of Bipolaris papendorfii: from phylogenetic to comparative genomic analysis

Bipolaris papendorfii has been reported as a fungal plant pathogen that rarely causes opportunistic infection in humans. Secondary metabolites isolated from this fungus possess medicinal and anticancer properties. However, its genetic fundamental and basic biology are largely unknown. In this study, we report the first draft genome sequence of B. papendorfii UM 226 isolated from the skin scraping of a patient. The assembled 33.4 Mb genome encodes 11,015 putative coding DNA sequences, of which, 2.49% are predicted transposable elements. Multilocus phylogenetic and phylogenomic analyses showed B. papendorfii UM 226 clustering with Curvularia species, apart from other plant pathogenic Bipolaris species. Its genomic features suggest that it is a heterothallic fungus with a putative unique gene encoding the LysM-containing protein which might be involved in fungal virulence on host plants, as well as a wide array of enzymes involved in carbohydrate metabolism, degradation of polysaccharides and lignin in the plant cell wall, secondary metabolite biosynthesis (including dimethylallyl tryptophan synthase, non-ribosomal peptide synthetase, polyketide synthase), the terpenoid pathway and the caffeine metabolism. This first genomic characterization of B. papendorfii provides the basis for further studies on its biology, pathogenicity and medicinal potential.     

The interactive effects of plant microbial symbionts: a review and meta-analysis

In nature, plants often associate with multiple symbionts concurrently, yet the effects of tripartite symbioses are not well understood. We expected synergistic growth responses from plants associating with functionally distinct symbionts. In contrast, symbionts providing similar benefits to a host may reduce host plant growth. We reviewed studies investigating the effect of multiple interactions on host plant performance. Additionally, we conducted a meta-analysis on the studies that performed controlled manipulations of the presence of two microbial symbionts. Using response ratios, we investigated the effects on plants of pairs of symbionts (mycorrhizal fungi, fungal endophytes, and nitrogen-fixers). The results did not support the view that arbuscular mycorrhizal (AM) fungi and rhizobia should interact synergistically. In contrast, we found the joint effects of fungal endophytes and arbuscular mycorrhizal fungi to be greater than expected given their independent effects. This increase in plant performance only held for antagonistic endophytes, whose negative effects were alleviated when in association with AM fungi, while the impact of beneficial endophytes was not altered by infection with AM fungi. Generalizations from the meta-analysis were limited by the substantial variation within types of interactions and the data available, highlighting the need for more research on a range of plant systems.     

Establishment of compatibility in the Ustilago maydis/maize pathosystem

The fungus Ustilago maydis is a biotrophic pathogen parasitizing on maize. The most prominent symptoms of the disease are large tumors in which fungal proliferation and spore differentiation occur. In this study, we have analyzed early and late tumor stages by confocal microscopy. We show that fungal differentiation occurs both within plant cells as well as in cavities where huge aggregates of fungal mycelium develop. U. maydis is poorly equipped with plant CWDEs and we demonstrate by array analysis that the respective genes follow distinct expression profiles at early and late stages of tumor development. For the set of three genes coding for pectinolytic enzymes, deletion mutants were generated by gene replacement. Neither single nor triple mutants were affected in pathogenic development. Based on our studies, we consider it unlikely that U. maydis feeds on carbohydrates derived from the digestion of plant cell wall material, but uses its set of plant CWDEs for softening the cell wall structure as a prerequisite for in planta growth.     

Unraveling the role of fungal symbionts in plant abiotic stress tolerance

Fungal symbionts have been found to be associated with every plant studied in the natural ecosystem, where they colonize and reside entirely or partially in the internal tissues of their host plant. Fungal endophytes can express/form a range of different lifestyle/relationships with different host including symbiotic, mutualistic, commensalistic and parasitic in response to host genotype and environmental factors. In mutualistic association fungal endophyte can enhance growth, increase reproductive success and confer biotic and abiotic stress tolerance to its host plant. Since abiotic stress such as, drought, high soil salinity, heat, cold, oxidative stress and heavy metal toxicity is the common adverse environmental conditions that affect and limit crop productivity worldwide. It may be a promising alternative strategy to exploit fungal endophytes to overcome the limitations to crop production brought by abiotic stress. There is an increasing interest in developing the potential biotechnological applications of fungal endophytes for improving plant stress tolerance and sustainable production of food crops. Here we have described the fungal symbioses, fungal symbionts and their role in abiotic stress tolerance. A putative mechanism of stress tolerance by symbionts has also been covered.Key words: abiotic stress, endophytes, fungal symbiont, mycorrhizal fungus, Piriformospora indica, stress tolerance, symbiosis     

Immunohistochemical investigation of the necrotrophic phase of the fungus Colletotrichum gloeosporioides in the biocontrol of hemp sesbania (Sesbania exaltata; Papilionaceae)

? Premise of the study: Fungal plant pathogens exert much of their effect on plant cells through alterations in the host cell walls. However, obtaining biochemical proof for this change is difficult because of the relatively small number of cells that are affected by the pathogen relative to the bulk of host tissue. In this study, we examined the differences in host wall composition between infected and uninfected areas of seedlings of the weed hemp sesbania (Sesbania exaltata) that were treated with the biocontrol agent Colletotrichum gloeosporioides. ? Methods: To determine the changes in cell wall composition, we used semi-thin sections and a battery of antibody probes that recognize components of the cell wall and immunogold-silver cytochemistry to visualize the probes. ? Key results: A loss of specific plant cell wall polysaccharides in the region surrounding the primary fungal infection and the creation of a defensive layer by the plant to limit the fungal invasion were the two most obvious changes noted in this study. At the invasion site, there was significant loss of rhamnogalacturon-1 (RGI) and esterified and de-esterified homogalacturonan (HG)-reactive epitopes from the cell walls. In contrast, boundary tissue between the vascular tissue and the fungal lesion reacted more strongly with antibodies that recognize arabinogalactan proteins (AGPs) and xyloglucans than in unaffected areas. ? Conclusions: These data strongly indicate a role of pectinases in the invasion of the biocontrol agent and the importance of extensins, AGPs, and xyloglucans as defense by the host.     

Wheat cells accumulate a syringyl-rich lignin during the hypersensitive resistance response

The stem rust fungus Puccinia graminis f.sp. tritici is an obligately biotrophic pathogen attacking wheat (Triticum aestivum). In compatible host/pathogen-interactions, the fungus participates in the host's metabolism by establishing functional haustoria in the susceptible plant cells. In highly resistant wheat cultivars, fungal attack is stopped by a hypersensitive response of penetrated host cells. This mechanism of programmed cell death of single plant cells is accompanied by the intracellular accumulation of material with UV-fluorescence typical of phenolic compounds. A similar reaction can be induced in healthy wheat leaves by the application of a rust-derived elicitor. We analysed the biochemical composition of this defense-induced phenolic material. Contents of total soluble and cell wall esterified and etherified phenolic acids were determined in rust-inoculated and elicitor-treated leaves of the fully susceptible wheat cultivar Prelude and its highly resistant, near-isogenic line Prelude-Sr5. While no resistance-related changes occured in any of these fractions, the lignin content as determined by the thioglycolic acid and the acetyl bromide methods increased after elicitor treatment. Nitrobenzene oxidation revealed that the entire increase can be explained by an increase in syringyl units only. These biochemical data were confirmed by fluorescence emission spectra analyses which indicated a defense-induced enrichment of syringyl lignin for cell wall samples both from elicitor-treated wheat leaves and single host cells undergoing a hypersensitive response upon fungal penetration.     

Fourier transform infrared-spectroscopic characterisation of isolated endodermal cell walls from plant roots: chemical nature in relation to anatomical development

The chemical nature of enzymatically isolated endodermal cell walls from Cicer arietinum L., Clivia miniata Reg. and Iris germanica L. was studied by FTIR (Fourier transform infrared) spectroscopy. Observed frequencies were assigned to functional groups present in the cell wall and relative amounts of the biopolymers suberin and lignin, cell wall carbohydrates and proteins were determined. Infrared absorption spectra indicated structural characteristics for the three different developmental states of the isolated endodermal cell wall: primary endodermis with Casparian strips (state I), secondary endodermis with suberin lamellae (state II), and tertiary endodermis with U-shaped cell wall depositions (state III). The data obtained from this study are compared with previous results obtained by chemical degradation of isolated endodermal cell walls and subsequent determination of monomeric degradation products by gas chromatography and mass spectrometry. It is concluded that FTIR spectroscopy represents a direct and nondestructive method suitable for the rapid investigation of isolated plant cell walls. Furthermore, the observation that the suberin-assigned absorption bands disappeared after transesterification of the samples with BF₃-methanol confirmed that suberin is completely degraded by this treatment. Received: 20 February 1999 / Accepted: 25 May 1999     

Establishment of compatibility in the Ustilago maydis/maize pathosystem

The fungus Ustilago maydis is a biotrophic pathogen parasitizing on maize. The most prominent symptoms of the disease are large tumors in which fungal proliferation and spore differentiation occur. In this study, we have analyzed early and late tumor stages by confocal microscopy. We show that fungal differentiation occurs both within plant cells as well as in cavities where huge aggregates of fungal mycelium develop. U. maydis is poorly equipped with plant CWDEs and we demonstrate by array analysis that the respective genes follow distinct expression profiles at early and late stages of tumor development. For the set of three genes coding for pectinolytic enzymes, deletion mutants were generated by gene replacement. Neither single nor triple mutants were affected in pathogenic development. Based on our studies, we consider it unlikely that U. maydis feeds on carbohydrates derived from the digestion of plant cell wall material, but uses its set of plant CWDEs for softening the cell wall structure as a prerequisite for in planta growth.     

Class V chitin synthase determines pathogenesis in the vascular wilt fungus Fusarium oxysporum and mediates resistance to plant defence compounds

Chitin, a beta-1,4-linked polysaccharide of N-acetylglucosamine, is a major structural component of fungal cell walls. Fungi have multiple classes of chitin synthases that catalyse N-acetylglucosamine polymerization. Here, we demonstrate the requirement for a class V chitin synthase during host infection by the vascular wilt pathogen Fusarium oxysporum. The chsV gene was identified in an insertional mutagenesis screen for pathogenicity mutants. ChsV has a putative myosin motor and a chitin synthase domain characteristic of class V chitin synthases. The chsV insertional mutant and a gene replacement mutant of F. oxysporum display morphological abnormalities such as hyphal swellings that are indicative of alterations in cell wall structure and can be partially restored by osmotic stabilizer. The mutants are unable to infect and colonize tomato plants or to grow invasively on tomato fruit tissue. They are also hypersensitive to plant antimicrobial defence compounds such as the tomato phytoanticipin alpha-tomatine or H2O2. Reintroduction of a functional chsV copy into the mutant restored the growth phenotype of the wild-type strain. These data suggest that F. oxysporum requires a specific class V chitin synthase for pathogenesis, most probably to protect itself against plant defence mechanisms.     

Characterization of major hydrolytic enzymes secreted by Pythium myriotylum, causative agent for soft rot disease

Pythium myriotylum, an oomycetous necrotroph is the causal agent of soft rot disease affecting several crops. Successful colonization by necrotrophs depends on their secretion of a diverse array of plant cell wall degrading enzymes (CWDEs). The induction dynamics of CWDEs secreted by P. myriotylum was analysed as little information is available for this pathogen. Activities of CWDEs that included pectinase, cellulase, xylanase and protease were detected using radial diffusion assay and differential staining. In Czapek Dox minimal medium supplemented with respective substrates as carbon source, the increase in CWDE activities was observed till 8 days of incubation after which a gradual decline in enzymatic activities was observed. With sucrose as sole carbon source, all the enzymes studied showed increase in activity with fungal growth while with cell wall material derived from ginger rhizome as sole carbon source, an initial spurt in cellulase, xylanase and pectinase activities was observed 3 days post incubation while protease activity increased from three days of incubation and reached maximum at 13 days of incubation. To further evaluate the role of CWDEs in pathogenicity, UV-induced mutants (pmN14uv1) were generated wherein significant reduction in cellulase, pectinase and protease activities were observed while that of xylanase remained unchanged compared to wild type isolate (RGCBN14). Bioassays indicated changes in infection potential of pmN14uv1 thereby suggesting the crucial role played by P. myriotylum CWDEs in initiating the rotting process. Hence appropriate strategies that target the production/activity of these secretory hydrolytic enzymes will help in reducing disease incidence/pathogen virulence.     

Ultrastructure of Tobacco Ringspot Virus-Induced Local Lesions in Lima Bean Leaves

Lignin is hypothesized to be a factor in the ability of cucurbits to resist fungal infection. To determine if lignin was present around sites of infection, susceptible cucumber plants and cucumber plants with systemic induced resistance were inoculated with Colletotrichum lagenarium, and the ultrastructure of plant and pathogen was examined 24 to 72 hours later. Conidia produced germ tubes with appressoria within 24 hours of inoculation on both control and induced plants. Penetration of plant epidermal cell walls occurred by 48 hours, and papillae formed at the site of penetration. Both elemental bromine and potassium permanganate were used to histochemically stain for lignin. Potassium permanganate, used together with energy dispersive X-ray microanalysis (EDS), indicated the presence of lignin in papillae and in electron-dense areas of plant cell walls directly beneath the appressoria. Using EDS, silicon was shown to be localized in the electron-dense areas of cucumber leaf cells. This is the first report of silicon in induced cucumber plants. There were no qualitative differences between the resistance reactions of induced and control plants.     

Constitutive expression of a fungal glucuronoyl esterase in Arabidopsis reveals altered cell wall composition and structure

A family 15 carbohydrate esterase (CE15) from the white‐rot basidiomycete, Phanerochaete carnosa (PcGCE), was transformed into Arabidopsis thaliana Col‐0 and was expressed from the constitutive cauliflower mosaic virus 35S promoter. Like other CE15 enzymes, PcGCE hydrolyzed methyl‐4‐O‐methyl‐d ‐glucopyranuronate and could target ester linkages that contribute to lignin–carbohydrate complexes that form in plant cell walls. Three independently transformed Arabidopsis lines were evaluated in terms of nine morphometric parameters, total sugar and lignin composition, cell wall anatomy, enzymatic saccharification and xylan extractability. The transgenic lines consistently displayed a leaf‐yellowing phenotype, as well as reduced glucose and xylose content by as much as 30% and 35%, respectively. Histological analysis revealed 50% reduction in cell wall thickness in the interfascicular fibres of transgenic plants, and FT‐IR microspectroscopy of interfascicular fibre walls indicated reduction in lignin cross‐linking in plants overexpressing PcGCE. Notably, these characteristics could be correlated with improved xylose recovery in transgenic plants, up to 15%. The current analysis represents the first example whereby a fungal glucuronoyl esterase is expressed in Arabidopsis and shows that the promotion of glucuronoyl esterase activity in plants can alter the extent of intermolecular cross‐linking within plant cell walls.     

A fungal endosymbiont affects host plant recruitment through seed- and litter-mediated mechanisms

1.  Many grass species are associated with maternally transmitted fungal endophytes. Increasing evidence shows that endophytes enhance host plant success under varied conditions, yet studies have rarely considered alternative mechanisms whereby these mutualistic symbionts may affect regeneration from seed.
2.  We performed a microcosm experiment to evaluate whether infection with Neotyphodium occultans affects recruitment in the annual grass Lolium multiflorum either directly, by infecting the seeds, or indirectly, by altering the suitability of recruitment microsites through the litter shed by host plants. Endophyte effects on establishment were tested for different litter depths and watering regimes under natural herbivory by leaf-cutting ants.
3.  Seed infection increased seedling emergence through the litter as well as final recruitment, irrespective of microsite conditions. However, litter produced by infected plants delayed emergence and decreased density of both infected and non-infected grass populations.
4.  Individual plant biomass did not change with seed infection but was increased under deep litter from endophyte-infected plants. Although seed infection did not protect establishing plants from leaf-cutting ants, herbivory was reduced in the presence of deep litter shed by infected plants.
5.  We conclude that fungal endophytes may affect host plant recruitment across subsequent generations not only by infecting the seeds but also through the host's dead remains. While the former effect entailed an advantage to infected plants, litter-mediated effects did not discriminate by infection status, and generally promoted the establishment of fewer and larger plants. Thus hidden foliar symbionts may play an underappreciated role in maintaining host species dominance through the litter produced by prior patch occupants.     

植物细胞壁介导的抗病性及其分子机制

本文简要介绍植物与病原菌在细胞壁层面上的相互作用,并从植物细胞对受侵过程中细胞壁损伤的感知、细胞壁损伤引起植物抗病信号途径的活化、植物细胞壁防卫反应的分子机制等方面重点概述植物细胞壁抗性及其分子机制。     

An ecological framework for understanding the roles of Epichloë endophytes on plant defenses against fungal diseases

Plants harbor a wide diversity of microorganisms in their tissues. Some of them have a long co-evolutionary history with their hosts, likely playing a pivotal role in regulating the plant interaction with other microbes such as pathogens. Some cool-season grasses are symbiotic with Epichloë fungal endophytes that grow symptomless and systemically in aboveground tissues. Among the many benefits that have been ascribed to endophytes, their role in mediating plant interactions with pathogens has been scarcely developed. Here, we explored the effects of Epichloë fungal endophytes on the interaction of host grasses with fungal pathogens. We made a meta-analysis that covered a total of 18 host grass species, 11 fungal endophyte species, and 22 fungal pathogen species. We observed endophyte-mediated negative effects on pathogens in vitro and in planta. Endophyte negative effects on pathogens were apparent not only in laboratory but also in greenhouse and field experiments. Epichloë fungal endophytes had negative effects on pathogen growth and spores' germination. On living plants, endophytes reduced both severity and incidence of the disease as well as colonization and subsequent infection of seeds. Symbiosis with endophytes showed an inhibitory effect on debilitator and killer pathogens, but not on castrators, and this effect did not differ among biotrophic or necrotrophic lifestyles. We found that this protection can be direct through the production of fungistatic compounds, the competition for a common resource, or the induction of plant defenses, and indirect associated with endophyte-generated changes in the abiotic or the biotic environment. Several mechanisms operate simultaneously and contribute differentially to the reduction of disease within grass populations.     

Secretome analysis of the phytopathogen Macrophomina phaseolina cultivated in liquid medium supplemented with and without soybean leaf infusion

Macrophomina phaseolina (Tassi) Goid. is a fungal pathogen that causes root and stem rot in several economically important crops. However, most of disease control strategies have shown limited effectiveness. Despite its impact on agriculture, molecular mechanisms involved in the interaction with host plant remains poorly understood. Nevertheless, it has been proven that fungal pathogens secrete a variety of proteins and metabolites to successfully infect their host plants. In this study, a proteomic analysis of proteins secreted by M. phaseolina in culture media supplemented with soybean leaf infusion was performed. A total of 250 proteins were identified with a predominance of hydrolytic enzymes. Plant cell wall degrading enzymes together peptidases were found, probably involved in the infection process. Predicted effector proteins were also found that could induce plant cell death or suppress plant immune response. Some of the putative effectors presented similarities to known fungal virulence factors. Expression analysis of ten selected protein-coding genes showed that these genes are induced during host tissue infection and suggested their participation in the infection process. The identification of secreted proteins of M. phaseolina could be used to improve the understanding of the biology and pathogenesis of this fungus. Although leaf infusion was able to induce changes at the proteome level, it is necessary to study the changes induced under conditions that mimic the natural infection process of the soil-borne pathogen M. phaseolina to identify virulence factors.     

Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis>

Background  

In order to initiate plant infection, fungal spores must germinate and penetrate into the host plant. Many fungal species differentiate specialized infection structures called appressoria on the host surface, which are essential for successful pathogenic development. In the model plant pathogen Magnaporthe grisea completion of mitosis and autophagy cell death of the spore are necessary for appressoria-mediated plant infection; blocking of mitosis prevents appressoria formation, and prevention of autophagy cell death results in non-functional appressoria.     

Lignin isolated from primary walls of hybrid aspen cell cultures indicates significant differences in lignin structure between primary and secondary cell wall.

Hybrid aspen (Populus tremula x tremuloides) cell cultures were grown for 7, 14 and 21 days. The cell cultures formed primary cell walls but no secondary cell wall according to carbohydrate analysis and microscopic characterization. The primary walls were lignified, increasingly with age, according to Klason lignin analysis. Presence of lignin in the primary walls, with a higher content in 21-day old cells than in 7-day old cells, was further supported by phloroglucinol/HCl reagent test and confocal microscopy after both immunolocalization and staining with acriflavin. Both laccase and peroxidase activity were found in the cultures and the activity increased during lignin formation. The lignin from the cell culture material was compared to lignin from mature aspen wood, where most of the lignin originates in the secondary cell wall, and which served as our secondary cell wall control. Lignin from the cell walls was isolated and characterized by thioacidolysis followed by gas chromatography and mass spectrometry. The lignin in the cell cultures differed from lignin of mature aspen wood in that it consisted exclusively of guaiacyl units, and had a more condensed structure. Five lignin structures were identified by mass spectrometry in the cell suspension cultures. The results indicate that the hybrid aspen cell culture used in this investigation may be a convenient experimental system for studies of primary cell wall lignin.