Gold-conjugated arabinogalactan-protein and other lectins as ultrastructural probes for the wheat/stem rust complex

Arabinogalactan-protein (AGP, "beta-lectin") was isolated from leek seeds, tested for specificity, conjugated with gold colloids, and used as a cytochemical probe to detect beta-linked bound sugars in ultrathin sections of wheat leaves infected with a compatible race of stem rust fungus. Similar sections were probed with other gold-labeled lectins to detect specific sugars. AGP-gold detected beta-glycosyl in all fungal walls and in the extrahaustorial matrix. Other lectin gold conjugates localized galactose in all fungal walls except in walls of the haustorial body. Limulus polyphemus lectin bound only to the outermost layer of intercellular hyphal walls of the fungus. Binding of these lectins was inhibited by their appropriate haptens and was diminished or abolished in specimens pretreated with protease, indicating that the target substances in the tissue were proteinaceous or that polysaccharides possessing affinity to the lectin probes had been removed by the enzyme from a proteinaceous matrix by passive escape. Binding of Lotus tetragonolobus lectin was limited to the two outermost fungal wall layers but was not hapten-inhibitable. Limax flavus lectin, specific for sialic acids, had no affinity to any structure in the sections. In the fungus, the most complex structure was the outermost wall layer of intercellular hyphal cells; it had affinity to all lectins tried so far, except to Limax flavus lectin and to wheat germ lectin included in an earlier study. In the host, AGP and the galactose-specific lectins bound to the inner domain of the wall in areas not in contact with the fungus. At host cell penetration sites, affinity to these lectins often extended throughout the host wall, confirming that it is modified at these sites. Pre-treatment with protease had no effect on lectin binding to the host wall. After protease treatment, host starch granules retained affinity to galactose-specific lectins, but lost affinity for AGP.     

Small oligomers of galacturonic acid are endogenous suppressors of disease resistance reactions in wheat leaves

Plants infected by a phytopathogenic fungus appear to recognize the presence of the pathogen by the molecular recognition of fungal cell wall fragments, termed 'elicitors', or of breakdown products of their own cell walls, termed 'endogenous elicitors'. Successful pathogens are thought to counteract this elicitation of active resistance reactions by the production of 'suppressors'. Evidence is presented here that fragments of the host cell wall, presumably produced enzymatically during fungal penetration, may act as 'endogenous suppressors' of resistance reactions in wheat. Pectic fractions were extracted from wheat cell walls by a variety of methods: Ca²⁺-chelators (CDTA and imidazole), a commercial mixture of pectic enzymes (Pectolyase Y23), a highly purified recombinant endopolygalacturonase (EPG), and solvolyses of the cell walls in anhydrous hydrogen fluoride at low temperatures followed by imidazole extraction. All of these fractions suppressed elicitor-induced activities of phenylalanine ammonia-lyase and peroxidases when co-injected with a glycoproteogalactan-elicitor, isolated from germ tubes of the wheat stem rust fungus, into the intercellular spaces of wheat leaves. Suppressor activity was correlated with the content of galacturonic acid in the extracts. Of the oligogalacturonides tested (monomer to hexamer), the dimer and trimer proved to be most active. This was not only true for suppression of elicitor-induced responses, but also for suppression of the hypersensitive resistance reaction in infected, genetically resistant host plants. As a consequence of reduced host cell necrosis in suppressor-treated leaves, the fungus developed larger colonies than in water-treated control leaves. Small oligomers of galacturonic acid, thus, are endogenous suppressors of resistance reactions in wheat leaves.     

条形柄锈菌(小麦条锈病菌)侵染结构体外形成的观察

通过荧光显微镜和扫描电镜分别对条形柄锈菌夏孢子在寄主植物-小麦叶表和非寄主植物-水稻叶表以及小麦穗部和茎秆上的萌发过程进行了观察。结果发现,夏孢子在小麦叶片体表萌发产生芽管后,可依次分化形成气孔下囊、初生菌丝与吸器母细胞;在小麦颖片、稃片及茎秆部位表面,同样可观察到病菌在体外分化形成吸器母细胞;并且在水稻叶片上也观察到病菌侵染结构存在体外分化现象。经荧光染色发现,条形柄锈菌在体外与在小麦组织中形成的侵染结构没有明显的差别。观察结果可为条形柄锈菌侵染结构的离体诱导与调控机理研究提供依据。     

Ultrastructural Observations on the Rust Fungus Puccinia malvacearum in Malva sylvestris ssp. mauritiana

Abstract: Our investigations show the structure of the intracellular mycelium of the rust fungus, Puccinia malvacearum, in leaves of Malva sylvestris ssp. mauritiana. Some intracellular structures of the fungus pass through, others end in the host cells. Fungal infection leads to an increase in the number of mucilage cells in the leaves; but mucilage production does not stop fungal growth. In contrast, the fungus is able to invade mucilage cells without any sign of fungal degradation.     

Extracellular beta-1,3-Glucanases in Stem Rust-Affected and Abiotically Stressed Wheat Leaves : Immunocytochemical Localization of the Enzyme and Detection of Multiple Forms in Gels by Activity Staining with Dye-Labeled Laminarin

Endo-β-1,3-glucanase activity in intercellular washing fluid (IWF) from leaves of wheat (Triticum aestivum) increased 10-fold 4 days after leaves were infected with the wheat stem rust fungus (Puccinia graminis f.sp. tritici), while exo-β-1,3-glucanase activity remained unchanged at a low level. Heat and ethylene stress had no effect, whereas mercury treatment resulted in a 2-fold increase in endo-β-1,3-glucanase activity. With a new method of activity staining using laminarin-Remazol brilliant blue as substrate in overlay gels, 18 electrophoretic forms of endo-β-1,3-glucanase were detected in IWF from unstressed leaves and up to 24 forms in IWF from stem rust-infected leaves. Most of the increase in β-1,3-glucanase activity and in the number of β-1,3-glucanases after rust infection was due to a nonspecific, stress-related effect on the plant, but two major forms of the enzyme probably originated from the fungus. β-1,3-Glucanase was localized cytochemically with anti-barley-β-1,3-glucanase antibodies. With preembedding labeling, the enzyme was demonstrated on the outside of host and fungal cell walls. Postembedding labeling localized the enzyme in the host plasmalemma and in the domain of host cell walls adjoining the plasmalemma, throughout walls of intercellular hyphal cells and haustoria, in the fungal cytoplasm, and in the extrahaustorial matrix. Cross-reactivity of β-1,3-glucanases from wheat and germinated uredospores of the rust fungus with the anti-barley-β-1,3-glucanase antibodies was confirmed in dot blot assays and on Western blots.     

Lectin Binding Studies on the Cell Walls of Soybean Rust (Phakopsora pachyrhizi Syd.)

Walls of uredospores, infection structures, intercellular hyphae and haustoria of the soybean rust fungus (Phakopsora pachyrhizi) were studied by electron microscopy using gold-labeled wheat germ lectin (WGL) and Concanavalin A (ConA) as cytochemical probes. Receptors for WGL (probably chitin) were detected in all fungal walls included in this study. WGL-binding occurred throughout the entire walls (uredospores, appressorial cone, penetration hyphae, haustorial mother cells) or only to the inner wall layers (germ tubes, appressoria, intercellular hyphae).     

Biological function and molecular mapping of M antigen in yeast phase of Histoplasma capsulatum

Histoplasmosis, due to the intracellular fungus Histoplasma capsulatum, can be diagnosed by demonstrating the presence of antibodies specific to the immunodominant M antigen. However, the role of this protein in the pathogenesis of histoplasmosis has not been elucidated. We sought to structurally and immunologically characterize the protein, determine yeast cell surface expression, and confirm catalase activity. A 3D-rendering of the M antigen by homology modeling revealed that the structures and domains closely resemble characterized fungal catalases. We generated monoclonal antibodies (mAbs) to the protein and determined that the M antigen is present on the yeast cell surface and in cell wall/cell membrane preparations. Similarly, we found that the majority of catalase activity was in extracts containing fungal surface antigens and that the M antigen is not significantly secreted by live yeast cells. The mAbs also identified unique epitopes on the M antigen. The localization of the M antigen to the cell surface of H. capsulatum yeast and the characterization of the protein's major epitopes have important implications since it demonstrates that although the protein may participate in protecting the fungus against oxidative stress it is also accessible to host immune cells and antibody.     

Cytological Study of Wheat Spike Infection by Bipolaris sorokiniana

The infection of wheat spikelets by Bipolaris sorokiniana , the causal agent of black point on grains and grain shrivelling, was examined by light and electron microscopy. Conidia of the pathogen germinated 6–12 h after inoculation on the surfaces of the different spike tissues. Extracellular sheaths were observed on germ tubes and appressoria attached to the surfaces of lemma, palea and seeds, but were only scarcely detected on the surface of conidia. Appressoria, frequently found over grooves, formed penetration hyphae invading the epidermal cell walls. Infection process was similar on the surface of the lemma, palea and glume. Growth of the fungus in the epidermal and parenchyma cells was found predominantly in the cell walls, and hyphae also extended intercellularly and intracellularly. Infection of seeds appeared to occur via two ways: (i) direct infection of the outer layers of the cell walls of the pericarp and (ii) through entering the stigma into the pericarp cells. Secretion of host cell wall hydrolytic enzymes at the apex of the penetrating hyphae may facilitate the spread of the fungus. In addition, toxins secreted by the fungus might explain the rapid death of host cells in contact with or distant to fungal cells. A host response to fungal infection involved the development of appositions between cell wall and plasma membrane in cells adjacent to fungal cells. Fungal hyphae were sometimes also surrounded by electron dense material.     

Use of green fluorescent protein to quantify the growth of Colletotrichum during infection of tobacco

To develop a quantitative assay of fungal growth inside plant tissues, strains of Colletotrichum destructivum and Colletotrichum orbiculare were transformed with a modified green fluorescent protein (GFP) gene fused with a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus nidulans. Transformants expressed GFP in culture and had the same growth rate and general appearance as the wild type. GFP was observed in all fungal structures during infection of leaves of Nicotiana benthamiana, except for the melanized appressoria and setae. The timing and appearance of the fungal structures in the host appeared to be identical to that of the wild type. GFP accumulation in inoculated leaves of N. benthamiana was quantified in leaf extracts using a fluorescence microplate reader, and the quantity of fluorescence was strongly correlated with the growth of the fungus as measured by the amount of fungal actin gene expression using Northern blot hybridizations. These results demonstrated that assaying green fluorescence levels from a GFP-transformed fungus is an accurate, fast and easy means of quantifying fungal growth inside host plant cells.     

The Differentiation of Infection Structures as a Result of Recognition Events between some Biotrophic Parasites and their Hosts

Most uredospores of rust fungi develop infection structures in a typical pattern so that they can infect the host plant. The function of these infection structures is divided into the following three phases:

• 1 In the recognition phase, the germ tube recognizes the cuticle and the stoma. This process may occur independently from the host plant since copies of the cuticle induce similar reactions of the fungus. During fungal growth on the epidermis, unspecific stress responses of the plant are triggered.
• 2 In the signal phase, the fungal substomatal vesicle and infection hypha(e) contact the host cells within the leaf parenchyma. A signal from the host induces further development of the fungus. Haustorium mother cell differentiation is effected and haustorium formation is initiated. At the same time, the fungus suppresses the synthesis of stress metabolites by the plant.
• 3 In the parasitic phase, the fungus penetrates the host cell and complex interactions between host and parasite begin. A highly specialized interface around the haustorium develops presumably in order to allow a more efficient nutrient transfer from host to parasite. Eventual defence reactions of the plant, generally on the race-cultivar level, fail to be evoked or are suppressed in compatible combinations.

     

Gold-conjugated arabinogalactan-protein and other lectins as ultrastructural probes for the wheat/stem rust complex

Summary Arabinogalactan-protein (AGP, -lectin) was isolated from leek seeds, tested for specificity, conjugated with gold colloids, and used as a cytochemical probe to detect -linked bound sugars in ultrathin sections of wheat leaves infected with a compatible race of stem rust fungus. Similar sections were probed with other gold-labeled lectins to detect specific sugars. AGP-gold detected -glycosyl in all fungal walls and in the extrahaustorial matrix. Other lectin gold conjugates localized galactose in all fungal walls except in walls of the haustorial body. Limulus polyphemus lectin bound only to the outermost layer of intercellular hyphal walls of the fungus. Binding of these lectins was inhibited by their appropriate haptens and was diminished or abolished in specimens pretreated with protease, indicating that the target substances in the tissue were proteinaceous or that polysaccharides possessing affinity to the lectin probes had been removed by the enzyme from a proteinaceous matrix by passive escape. Bindig of Lotus tetragonolobus lectin was limited to the two outermost fungal wall layers but was not hapten-inhibitable. Limax flavus lectin, specific for sialic acids, had no affinity to any structure in the sections. In the fungus, the most complex structure was the outermost wall layer of intercellular hyphal cells; it had affinity to all lectins tried so far, except to Limax flavus lectin and to wheat germ lectin included in an earlier study. In the host, AGP and the galactose-specific lectins bound to the inner domain of the wall in areas not in contact with the fungus. At host cell penetration sites, affinity to these lectins often extended througout the host wall, confirming that it is modified at these sites. Pre-treatment with protease had no effect on lectin binding to the host wall. After protease treatment, host starch granules retained affinity to galactose-specific lectins, but lost affinity for AGP.This paper is listed as Contribution No. 1330, Agriculture Canada Research Station Winnipeg     

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.     

Fusion of a novel genetically engineered chitosan affinity protein and green fluorescent protein for specific detection of chitosan in vitro and in situ

Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan-e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His(6) tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His(5) antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining.     

A Light and Electron Microscope Study of the Interaction of Yellow Rust (Puccinia striiformis) with a Susceptible Wheat Cultivar

The microscopy and ultrastructure of the interaction of Pucciniastriiformis with a susceptible wheat cultivar was examined atintervals from the time of first haustorium formation to theonset of sporulation. At any particular point in the radiallyexpanding area of infection a sequence of morphological changesoccurred in the infected host cells and the fungus which werecorrelated with successive phases of active fungal growth, accumulationof reserves and finally export of reserves to the developingreproductive structures. The observations are compared withprevious work on other host-rust interactions. yellow rust, Puccinia striiformis, wheat, host-pathogen interaction     

Uvitex2B: a rapid and efficient stain for detection of arbuscular mycorrhizal fungi within plant roots

The study of arbuscular mycorrhiza often requires the staining of fungal structures using specific dyes. Fluorescent dyes such as acid fuchsin and wheat germ agglutinin conjugates give excellent results, but these compounds are either hazardous or very expensive. Here, we show that a safer and inexpensive dye, Uvitex2B, can be efficiently used to stain intraradical fungal structures formed by the arbuscular mycorrhizal fungus Glomus intraradices in three plant species: carrot, Casuarina equisetifolia, and Medicago truncatula. The intensity and stability of Uvitex2B allow the acquisition of high-quality images using not only confocal laser scanning microscopy but also epifluorescence microscopy coupled with image deconvolution. Furthermore, we demonstrate that Uvitex2B and β-glucuronidase staining are compatible and can thus be used to reveal arbuscular mycorrhizal structures in the context of promoter activation analysis.     

Development of Early Infection Structures of Puccinia recondita f. sp. tritici in Non-host Cereal Species

The development of infection structures, derived from urediospores of Puccinia recondita f.sp. trilici in nearisogenic lines of susceptible and resistant wheat, and in non-hosts (viz. maize, oat, sorghum and barley), was examined by fluorescence microscopy and scanning electron microscopy (SEM). The infection structure formation on and in five cereal species follows a similar pattern. In sorghum, fungal development is arrested at the stage of substomatal vesicle formation, while, in maize, most fungal structures collapse during the stage of primary hypha development. By contrast, in wheat, barley and oat, the fungus forms many branched infection hyphae and haustorial mother cells.     

Enzymatic detachment of spore germlings in Magnaporthe oryzae

Magnaporthe oryzae germlings tightly attach to the host surface by producing extracellular matrix (ECM) from germ tubes and appressoria, which are important for the early infection process. To understand the adhesion mechanisms of ECM during differentiation of infection structure, we evaluated the effects of various enzymes on M. oryzae germlings and the disease symptoms of the host plant, wheat. Treatment with β-mannosidase, collagenase N-2, collagenase S-1, or gelatinase B at 1-h postinoculation (hpi) resulted in germling detachment, although producing normal appressoria. Treatment with matrix metalloproteinases (MMPs) at 6 hpi also caused germling detachment. Furthermore, we confirmed by the inoculation tests and scanning electron microscopy that the germlings on the wheat plant were removed and did not manifest pathogenicity on treatment with MMPs. The most effective MMPs were crude collagenase, collagenase S-1, and gelatinase B, suggesting that the application of MMPs is promising for crop protection from fungal diseases by its detachment action.