sRNA‐FISH: versatile fluorescent in situ detection of small RNAs in plants

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi‐photon fluorescence excitation microscopy can be used to separate the target sRNA‐FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level.     

Localisation of fungal hyphae in wood using immunofluorescence labelling and confocal laser scanning microscopy

This study explored the feasibility of using immunofluorescence labelling in conjunction with confocal laser scanning microscopy (CLSM) for detection of common fungal colonisers of unseasoned radiata pine in New Zealand. Wood sections infected with Ophiostoma piceae were treated with monoclonal antibody IF3 (1), and then Oregon green 514 goat anti-mouse IgG, a fluorescent secondary antibody. Additional wood sections infected with other Ophiostoma spp., Sphaeropsis sapinea, Leptographium procerum, Trichoderma sp. and Phlebiopsis gigantea were treated similarly to determine whether the antibody was specific to O. piceae or was recognising other fungal species. Sections were examined using phase contrast and fluorescence light microscopy prior to CLSM. Immunolabelled fungal hyphae showed relatively weak fluorescence compared to the strong autofluorescence of wood cell walls and extractives. Labelled hyphae of O. piceae were detected in wood using CLSM but not with ordinary fluorescence microscopy. This is because CLSM has stronger illumination power and superior imaging ability compared with ordinary fluorescence microscopy. The monoclonal antibody did not cross-react with the other Ophiostoma species. However, non-specific antibody binding was observed with L. procerum and Trichoderma species. Furthermore, cell walls of L. procerum showed strong autofluorescence with optical properties similar to wood extractives when examined prior to incubation with the monoclonal and secondary antibody, therefore cross-reactivity tests were inconclusive for Leptographium and Trichoderma species. The current investigation demonstrated that CLSM provides possibilities for future investigations on in situ interactions of common radiata pine fungal colonisers, with one another and with wood.     

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.     

Cytoskeleton in mycorrhizal symbiosis

An understanding of the role played by the cytoskeleton in formation and function of mycorrhizas has been hampered by the technical difficulty of working with mycorrhizal material. Recently, however, improved labelling techniques suitable for both plant and fungal symbionts in combination with either epifluorescence microscopy or laser scanning confocal microscopy have resulted in new information. As well, molecular methods have made it possible to monitor changes of cytoskeletal elements during mycorrhiza development. Currently we know that the cytoskeletal systems of both plant and fungal partners undergo changes during both ecto- and endomycorrhizal symbiosis. However, little information is available concerning the regulatory factors or the cause and effect relationship of cytoskeletal changes and cellular events. In this article, research involving the cytoskeleton of mycorrhizas is reviewed in detail, whereas basic information of the cytoskeleton of plant and fungal cells is only briefly discussed as background. A brief comparison is also made between the information on mycorrhizas with that of biotrophic pathogenic fungi and the Rhizobium–legume symbiosis.     

Autofluorescence detection of arbuscular mycorrhizal fungal structures in palm roots: an underestimated experimental method

The aim of this study was to reassess the use of autofluorescence for evaluating AM colonization in mycorrhizal roots in the light of criticisms of this method that affirmed that only metabolically inactive arbuscules autofluoresce. It was also investigated whether other mycorrhizal structures, such as hyphae, vesicles and spores, could be detected by autofluorescence, and whether the autofluorescence pattern of AM fungal structures could be exploited methodologically, for example, in the detection and sorting of spores by flow cytometry. Mycorrhizal roots of the palm species Brahea armata, Chamaerops humilis, Phoenix canariensis and Phoenix dactylifera were sectioned and observed by means of fluorescence microscopy. In addition, fungal structures isolated from mycorrhizal roots of P. dactylifera were examined. The same root sections and isolated fungal structures were subjected to vital staining with nitro blue tetrazolium to determine their metabolic state (active or inactive). Moreover, spores of Glomus intraradices, and Glomus clarum were studied by epifluorescence and flow cytometry. Mycorrhizal whole roots of Medicago sativa were also assessed by autofluorescence detection. In contrast to previous reports, the results presented in this paper clearly demonstrate that all fungal structures, both intra- and extraradical, autofluoresced under blue light excitation, regardless of their state (dead or alive). Some arbuscules isolated from roots and mature spores showed further autofluorescence under green light excitation. The source of the autofluorescence was localized in the fungal cell wall. It was shown that AM spores can be detected by flow cytometry. The results support the use of autofluorescence for the evaluation of AM colonization, at least in palm species, and refute previous criticisms of the method.     

Quantification of morphochemical changes during in situ enzymatic hydrolysis of individual biomass particles based on autofluorescence imaging

Enzymatic hydrolysis of biomass is an established method for producing biofuels. Lignocellulosic biomass such as corn stover is very inhomogeneous material with big variation on conversion rates between individual particles therefore leading to variable recalcitrance results. In this study, we used noninvasive optical microscopy techniques, such as two-photon microscopy and fluorescence lifetime imaging microscopy, to visualize and analyze morphological and chemical changes of individual corn stover particles pretreated with sulfuric acid during hydrolysis. Morphochemical changes were interpreted based on the fluorescence properties of isolated building blocks of plant cell wall, such as cellulose, hemicellulose, and lignin. Enzymatic hydrolysis resulted in particle size reduction, side wall collapse, decrease of second harmonic signal from cellulose, redshifting of autofluorescence emission, and lifetime decrease attributed to the relative increase of lignin. Based on these observations, tracking compositional change after hydrolysis of individual particles was accomplished. The methodologies developed offer a paradigm for imaging and analyzing enzymatic hydrolysis in vitro and in situ, which could be used for screening enzymes cocktails targeting specific recalcitrant structures or investigating locally enzyme anti-inhibitory agents.     

Colorimetric in situ hybridization (CISH) with digoxigenin-labeled oligonucleotide probes in autofluorescent hyphomycetes.

We used digoxigenin-labeled probes for in situ hybridization of hyphomycetes to replace the commonly used fluorescent proof of probe binding by a colorimetric reaction. The resulting blue-purple, intracellular precipitate could be easily detected by light microscopy, and thus presented a promising method to overcome autofluorescence of fungal material and substratum. Optimal cell fixation and permeabilization procedures, as well as hybridization conditions were developed on the example of two different hyphomycetes: Phialophora sp. and Cartapip, a colorless mutant of Ophiostoma piliferum (Agra Sol).     

Combining microtomy and confocal laser scanning microscopy for structural analyses of plant–fungus associations

The serious problem of extended tissue thickness in the analysis of plant–fungus associations was overcome using a new method that combines physical and optical sectioning of the resin-embedded sample by microtomy and confocal microscopy. Improved tissue infiltration of the fungal-specific, high molecular weight fluorescent probe wheat germ agglutinin conjugated to Alexa Fluor® 633 resulted in high fungus-specific fluorescence even in deeper tissue sections. If autofluorescence was insufficient, additional counterstaining with Calcofluor White M2R or propidium iodide was applied in order to visualise the host plant tissues. Alternatively, the non-specific fluorochrome acid fuchsine was used for rapid staining of both, the plant and the fungal cells. The intricate spatial arrangements of the plant and fungal cells were preserved by immobilization in the hydrophilic resin Unicryl?. Microtomy was used to section the resin-embedded roots or leaves until the desired plane was reached. The data sets generated by confocal laser scanning microscopy of the remaining resin stubs allowed the precise spatial reconstruction of complex structures in the plant–fungus associations of interest. This approach was successfully tested on tissues from ectomycorrhiza (Betula pendula), arbuscular mycorrhiza (Galium aparine; Polygala paniculata, Polygala rupestris), ericoid mycorrhiza (Calluna vulgaris), orchid mycorrhiza (Limodorum abortivum, Serapias parviflora) and on one leaf–fungus association (Zymoseptoria tritici on Triticum aestivum). The method provides an efficient visualisation protocol applicable with a wide range of plant–fungus symbioses.     

Reversible cytoplasmic rearrangements precede wall apposition,hypersensitive cell death and defense-related gene activation in potato/Phytophthora infestans interactions

We used video microscopy techniques as a tool for live examination of the dynamic aspects of plant/fungus interactions. Early, dynamic responses of epidermal midrib cells of leaves from a potato cultivar (Solanum tuberosum L. cv. Datura) carrying resistance gene R1 to Phytophthora infestans (race 1: compatible interaction, race 4: incompatible interaction) were monitored. Similar responses were observed in both types of interaction, ranging from no visible reaction of invaded plant cells to hypersensitive cell death. The overall defense response of each individual cell exhibited a highly dynamic behavior that appeared to be tightly coordinated with the growth of the fungus. Initial localized reactions, including major rearrangements within the cytoplasm, occurred directly at the fungal penetration site, where rapid apposition of autofluorescent material and callose took place. If fungal invasion stopped at this stage, the host cell restored its normal cytoplasmic activity and survived. Hypersensitive cell death occurred only when fungal growth had proceeded to the formation of a clearly identifiable haustorium. In such cases, cytoplasm and nucleus conglomerated around the intracellular fungal structure, followed by a sudden collapse of the whole conglomerate and an instantaneous collapse of the fungal haustorium. Only small quantitative differences between the compatible and incompatible interactions of the two fungal races were observed for these early responses of epidermal cells. In the incompatible interaction, a slightly larger number of epidermal cells responded to fungal attack. More pronounced quantitative differences between compatible and incompatible interactions occurred upon fungal invasion of the mesophyll. These differences in the number of responding cells were not reflected at the level of gene expression: the spatial and temporal activation patterns of two defense-related genes, encoding phenylalanine ammonia-lyase and pathogenesis-related protein 1, were similar in both types of interaction.Dedicated to Professor Peter Sitte, Freiburg, Germany, on the occasion of his 65th birthday     

Simultaneous specific in planta visualization of root-colonizing fungi using fluorescence in situ hybridization (FISH)

In planta detection of mutualistic, endophytic, and pathogenic fungi commonly colonizing roots and other plant organs is not a routine task. We aimed to use fluorescence in situ hybridization (FISH) for simultaneous specific detection of different fungi colonizing the same tissue. We have adapted ribosomal RNA (rRNA) FISH for visualization of common mycorrhizal (arbuscular- and ectomycorrhiza) and endophytic fungi within roots of different plant species. Beside general probes, we designed and used specific ones hybridizing to the large subunit of rRNA with fluorescent dyes chosen to avoid or reduce the interference with the autofluorescence of plant tissues. We report here an optimized efficient protocol of rRNA FISH and the use of both epifluorescence and confocal laser scanning microscopy for simultaneous specific differential detection of those fungi colonizing the same root. The method could be applied for the characterization of other plant–fungal interactions, too. In planta FISH with specific probes labeled with appropriate fluorescent dyes could be used not only in basic research but to detect plant colonizing pathogenic fungi in their latent life-period.     

Investigation on the infection mechanism of the fungus <Emphasis Type="Italic">Clonostachys rosea</Emphasis> against nematodes using the green fluorescent protein

The fungus Clonostachys rosea (syn. Gliocladium roseum) is a potential biocontrol agent. It can suppress the sporulation of the plant pathogenic fungus Botrytis cinerea and kill pathogenic nematodes, but the process of nematode pathogenesis is poorly understood. To help understand the underlying mechanism, we constructed recombinant strains containing a plasmid with both the enhanced green fluorescent protein gene egfp and the hygromycin resistance gene hph. Expression of the green fluorescent protein (GFP) was monitored using fluorescence microscopy. Our observations reveal that the pathogenesis started from the adherence of conidia to nematode cuticle for germination, followed by the penetration of germ tubes into the nematode body and subsequent death and degradation of the nematodes. These are the first findings on the infection process of the fungal pathogen marked with GFP, and the developed method can become an important tool for studying the molecular mechanisms of nematode infection by C. rosea. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lin Zhang and Jinkui Yang contributed equally to this work.     

<Emphasis Type="Italic">Bellis perennis</Emphasis>: a useful tool for protein localization studies

Fluorescent fusion proteins together with transient transformation techniques are commonly used to investigate intracellular protein localisation in vivo. Biolistic transfection is reliable, efficient and avoids experimental problems associated with producing and handling fragile protoplasts. Onion epidermis pavement cells are frequently used with this technique, their excellent properties for microscopy resulting from their easy removal from the underlying tissues and large size. They also have advantages over mesophyll cells for fluorescence microscopy, as they are devoid of chloroplasts whose autofluorescence can pose problems. The arrested plastid development is peculiar to epidermal cells, however, and stands in the way of studies on protein targeting to plastids. We have developed a system enabling studies of in vivo protein targeting to organelles including chloroplasts within a photosynthetically active plant cell with excellent optical properties using a transient transformation procedure. We established biolistic transfection in epidermal pavement cells of the lawn daisy (Bellis perennis L., cultivar “Galaxy red”) which unusually contain a moderate number of functional chloroplasts. These cells are excellent objects for fluorescence microscopy using current reporters, combining the advantages of the ease of biolistic transfection, the excellent optical properties of a single cell layer and access to chloroplast protein targeting. We demonstrate chloroplast targeting of plastid-localised heme oxygenase, and two further proteins whose localisation was equivocal. We also demonstrate unambiguous targeting to mitochondria, peroxisomes and nuclei. We thus propose that the Bellis system represents a valuable tool for protein localisation studies in living plant cells.     

Cellular interactions between arbuscular mycorrhizal fungi and rhizosphere bacteria

Summary We have investigated whether direct physical interactions occur between arbuscular mycorrhizal (AM) fungi and plant growth promoting rhizobacteria (PGPRs), some of which are used as biocontrol agents. Attachment of rhizobia and pseudomonads to the spores and fungal mycelium ofGigaspora margarita has been assessed in vitro and visualized by a combination of electron and confocal microscopy. The results showed that both rhizobia and pseudomonads adhere to spores and hyphae of AM fungi germinated under sterile conditions, although the degree of attachment depended upon the strain.Pseudomonas fluorescens strain WCS 365 andRhizobium leguminosarum strains B556 and 3841 were the most effective colonizers. Extracellular material of bacterial origin containing cellulose produced around the attached bacteria may mediate fungal/bacterial interactions. These results suggest that antagonistic and synergistic interactions between AM fungi and rhizosphere bacteria may be mediated by soluble factors or physical contact. They also support the view that AM fungi are a vehicle for the colonization of plant roots by soil rhizobacteria.Abbreviations AM arbuscular mycorrhiza - PGPR plant growth promoting rhizobacteria - CBH cellobiohydrolase - DAPG 2,4-(diacetyl-phloroglucinol - TY triptone-yeast - LB Lauria-Bertani Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Time-gated in vivo autofluorescence imaging of dental caries.

Laser-induced time-resolved autofluorescence from carious lesions of human teeth was studied by means of ultrashort pulsed laser systems, time-correlated single photon counting and time-gated imaging. Carious regions exhibited a slower fluorescence decay with a main 17 ns fluorescence lifetime than healthy hard dental tissue. The long-lived fluorophore present in carious lesions only emits in the red spectral region. Fluorescence decay time and spectral characteristics are typical of fluorescent metal-free porphyrin monomers. The spatial distribution of the long-lived endogenous porphyrin fluorophore within the tooth material was detected by time-gated nanosecond autofluorescence imaging. In particular, high contrast video images were obtained with an appropriate time delay of 15 ns to 25 ns between excitation and detection due to the suppression of short-lived autofluorescence of healthy tissue. First in vivo applications are reported indicating the potential of time-resolved fluorescence diagnostics for early caries- and dental plaque detection.     

Reef coral fluorescent proteins for visualizing fungal pathogens

The fluorescent proteins AmCyan, ZsGreen, ZsYellow, and AsRed, modified versions of proteins identified recently from several Anthozoa species of reef corals, were expressed for the first time in a heterologous system and used for imaging two different fungal plant pathogens. When driven by strong constitutive fungal promotors, expression of these reef coral fluorescent proteins yielded bright cytoplasmic fluorescence in Fusarium verticillioides and Magnaporthe grisea, and had no detectable effect on either growth rate or the ability to cause disease. Differential intracellular localization of the fluorescent proteins resulted in the discrimination of many subcellular organelles by confocal and multi-photon microscopy, and facilitated monitoring of such details as organelle dynamics and changes in the permeability of the nuclear envelope. AmCyan and ZsGreen were sufficiently excited at 855 and 880 nm, respectively, to allow for time-resolved in planta imaging by two-photon microscopy.     

Optimization of the HyPer sensor for robust real‐time detection of hydrogen peroxide in the rice blast fungus

Reactive oxygen species (ROS) production and breakdown have been studied in detail in plant‐pathogenic fungi, including the rice blast fungus, Magnaporthe oryzae; however, the examination of the dynamic process of ROS production in real time has proven to be challenging. We resynthesized an existing ROS sensor, called HyPer, to exhibit optimized codon bias for fungi, specifically Neurospora crassa, and used a combination of microscopy and plate reader assays to determine whether this construct could detect changes in fungal ROS during the plant infection process. Using confocal microscopy, we were able to visualize fluctuating ROS levels during the formation of an appressorium on an artificial hydrophobic surface, as well as during infection on host leaves. Using the plate reader, we were able to ascertain measurements of hydrogen peroxide (H₂O₂) levels in conidia as detected by the MoHyPer sensor. Overall, by the optimization of codon usage for N. crassa and related fungal genomes, the MoHyPer sensor can be used as a robust, dynamic and powerful tool to both monitor and quantify H₂O₂ dynamics in real time during important stages of the plant infection process.     

Mycoparasitic relationships

The mode of attack and the infection structures of the necrotrophic mycoparasite, Pythium acanthicum, as well as the responses of various fungal hosts to parasitism were studied using both electron and light microscopy. Many taxonomically distinct fungal hosts were used, though Phycomyces blakesleeanus, Pythium aphanidermatum, Rhizoctonia solani and a basidiomycete identified as Corticium sensu lato were studied in greatest detail. Parasitism was by direct penetration of the fungal host without appressorium formation by the parasite. The host's cells responded to contact by P. acanthicum by forming papillae. The morphological features of the papillae varied with the particular host. In P. blakesleeanus they were comprised of vesicles and segments of cytoplasm entrapped in a fibrillo-granular matrix, while in R. solani and the Corticium basidiomycete they contained considerable amounts of electron-opaque and electron-translucent material. Evidence for both mechanical and enzymatic penetration of the host fungi by the parasite are presented. Details of host wall and septum penetration by the parasite are presented using time-lapse light microscopy with in vivo systems. Many of these stages of parasitism were examined ultrastructurally. Some comparisons of these mycoparasitic relationships are discussed in relation to what is known from the literature about phytoparasitic interactions.     

Time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization.

Fluorescent lanthanide chelates with long decay times allow the suppression of the fast decaying autofluorescence in biological specimens. This property makes lanthanide chelates attractive as labels for fluorescence microscopy. As a consequence of the suppression of the background fluorescence the sensitivity can be increased. We modified a standard epifluorescence microscope for time-resolved fluorescence imaging by adding a pulsed light source and a chopper in the narrow aperture plane. A cooled CCD-camera was used for detection and the images were digitally processed. A fluorescent europium chelate was conjugated to antisera and to streptavidin. These conjugates were used for the localization of tumor associated antigen C242 in the malignant mucosa of human colon, for the localization of type II collagen mRNA in developing human cartilaginary growth plates, and for the detection of HPV type specific gene sequences in the squamous epithelium of human cervix. The specific slowly decaying fluorescence of the europium label could be effectively separated from the fast decaying background fluorescence. It was possible to use the europium label at the cell and tissue level and the autofluorescence was effectively suppressed in in situ hybridization and immunohistochemical reactions in both frozen and formaldehyde-fixed, wax-embedded specimens.     

两种混合样品策略对揭示杜鹃花根部真菌多样性的影响

由于土壤微生物群落物种组成的高度空间异质性,混合样品(sample pooling)被广泛应用于微生物多样性与群落结构研究。在根部真菌的分子检测中,样品混合策略以及测序的克隆数或序列数均对揭示真菌群落结构的准确性有影响。【目的】为建立一套能快速准确地反映杜鹃花属植物根部真菌的物种组成与群落结构的分子检测技术平台,【方法】本研究采集锈红杜鹃和亮鳞杜鹃多份根系样品分别提取DNA,比较PCR扩增前和扩增后混合策略构建的克隆文库中真菌物种组成的差异。【结果】在2种宿主植物根系中,多份样品在PCR扩增后混合构建的克隆文库检测到的根部真菌物种丰富度、真菌群落的Shannon-Wiener多样性指数均高于扩增前混合的克隆文库。高频度的根部真菌在2种克隆文库中均检测到,但低频度的真菌物种组成在2种克隆文库中完全不同。更重要的是,当采用广泛应用的真菌通用引物ITS1f和ITS4扩增根部真菌ITS序列时,PCR扩增后混合的方法能有效地减轻杜鹃花属植物ITS序列被优先扩增的现象。真菌物种累积曲线显示,当测序的真菌ITS片段克隆数达到50个左右,即能较全面地反映2种杜鹃花根部真菌物种组成。【结论】独立扩增多份根系样品DNA,再将PCR产物混合构建克隆文库的方法能更全面地揭示杜鹃花属植物根部真菌物种丰富度与物种组成。     

Genetic manipulation in vesicular-arbuscular mycorrhizal fungi

The symbiosis between vesicular-arbuscular mycorrhizal (VAM) fungi and host plants develops after successful interactions between both partners. These interactions probably involve signal molecules produced by the host plant, by the fungi, or by both. So far the biotrophic status of VAM fungi has hampered the understanding of the processes regulating their physiology. However, among different methods for co-cultivating VAM fungi, root organ cultures (ROC) appear to be a useful technique for studying VAM development. This system has been useful in defining the nutritional requirements of VAM fungi in the precolonization stage and in obtaining axenic fungal material in various developmental stages. The work discussed here focuses on the application of Polymerase Chain Reaction (PCR) technology and the potential of promoting hyphal growth in the absence of the plant. These techniques are being used to study VAM fungi in two main areas. The first concerns the determination of the DNA sequences coding for the SSU ribosomal RNA of two VAM fungi. This approach has allowed the design of specific primers for the rapid identification and quantification of VAM fungi. The second area of research concerns the potential use of PCR technology to study selective expression of specific genes during fungal spore development in defined in vitro conditions. The achievement of this future prospect depends on the ability to prepare PCR-based cDNA libraries from small amounts of fungal material after stimulation of hyphal growth with CO₂ and plant flavonols.