Ultrastructural characterisation of the host–pathogen interface in white blister-infected Arabidopsis leaves

In this study transmission electron microscopy (TEM) was used to examine details of the host–pathogen interface in Arabidopsis thaliana cotyledons infected by Albugo candida, causal agent of white blister. After successful entry through stomatal pores, the pathogen developed a substomatal vesicle and subsequently produced intercellular hyphae. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were spherical and 4.5 μm in diameter. Each haustorium was connected to intercellular hyphae by a narrow, slender haustorium neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. No obvious response was observed in host cells following formation of haustoria. Most of the mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cells suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotrophic Oomycete differs considerably from responses to other pathogens such as necrotrophs. Modification of the host plasma membrane (PM) along the cell wall and around the haustoria, was detected by applying the periodic acid-chromic acid-phosphotungstic acid (PACP) staining technique. After staining with PACP, the host PM was found to be intensely electron dense where it was adjacent to the host cell wall and the distal region of the haustorial neck. By contrast, the extrahaustorial membrane, where the host PM surrounded the haustorium, was consistently very lightly stained.     

Ultrastructural comparison of incompatible and compatible interactions in the barley powdery mildew disease

Summary In the powdery mildew disease of barley,Erysiphe graminis f. sp.hordei forms an intimate relationship with compatible hosts, in which haustoria form in epidermal cells with no obvious detrimental effects on the host until late in the infection sequence. In incompatible interactions, by contrast, the deposition of papillae and localized host cell death have been correlated with the cessation of growth byE. g. hordei. With the advent of improved, low temperature methods of sample preparation, we felt that it was useful to reevaluate the structural details of interactions between barley andE. g. hordei by transmission electron microscopy. The haustoria that develop in susceptible barley lines appear highly metabolically active based on the occurrrence of abundant endoplasmic reticulum, Golgi-like cisternae, and vesicles. In comparison, haustoria found in the resistant barley line exhibited varying signs of degradation. A striking clearing of the matrix and loss of cristae were typical early changes in the haustorial mitochondria in incompatible interactions. The absence of distinct endoplasmic reticulum and Golgi-like cisternae, the formation of vacuoles, and the occurrence of a distended sheath were characteristic of intermediate stages of haustorial degeneration. At more advanced stages of degeneration, haustoria were dominated by large vacuoles containing membrane fragments. This process of degeneration was not observed in haustoria ofE. g. hordei developing in the susceptible barley line.Abbreviations b endoplasmic reticulum extension, blebbing - er endoplasmic reticulum - f fibrillar material - g Golgi-like structure - h haustorium - hb haustorial body - hcw haustorial cell wall - hcy haustorial cytoplasm - hf haustorial finger - hocw host cell wall - hocy host cytoplasm - 1 lipid-like droplet - m mitochondrion - mt microtubule - mve multivesicular body - n nucleus - p papilla - ph penetration site of an infection peg - pl plasma membrane - s sheath - sm extrahaustorial membrane - v vacuole - ve vesicle     

Studies on new types of haustoria in some Central EuropeanRhinanthoideae (Scrophulariaceae)

Two new types of haustoria have been found in many representatives ofRhinanthoideae. Metahaustoria are stimulated by host roots but lack contact with them. They develop a massive haustorial core with a large- and a smalled-celled portion, but little vascular core. In very old metahaustoria the vascular core is much enlarged and numerous tracheal elements develop in the reduced haustorial core and cortex. Warthaustoria are very small, occur only in the upper portions of the root system, and have host contacts. Vascular and haustorial core, and the endophyte are lacking, the intrusive cells are usually not endogenous, but rather roothair-like. Wart-haustoria may represent reduced and partly inhibited normal haustoria.     

Molecular differentiation in pea powdery-mildew haustoria

Monoclonal antibodies have been raised against haustorial complexes isolated from pea (Pisum sativum L.) leaves infected by the biotrophic powdery mildew fungus Erysiphe pisi D.C. Immuno-localisation studies, using isolated haustorial complexes and infected pea leaf material, have shown that one of the antibodies, designated UB7, binds to fungal wall and plasma membranes present in both haustoria and mycelia. However, a second antibody, UB8, binds specifically to the haustorial plasma membrane, and does not label fungal plasma membranes in mycelia. Western blotting and antigen-modification techniques have shown that UB8 recognises a protein epitope of a 62-kDa antigen. A reduction in molecular weight of this component after endo-F treatment indicates that the antigen is an N-linked glycoprotein. UB7 also recognises a 62-kDa glycoprotein, which is susceptible to endo-F treatment, and the antibody binds to a carbohydrate epitope. Differences in molecular weights of the products after endo-F treatment of antigens show that the 62-kDa glycoproteins recognised by the antibodies are distinct molecules, in accordance with the localisation results. Overall, the results provide evidence for molecular differentiation associated with the development of haustoria in a biotrophic infection.Abbreviations ehm extrahaustorial membrane - ELISA enzyme-linked immunosorbent assay - HC haustorial complex - hpm haustorial plasma membrane - IIF indirect immunofluorescence - MAb monoclonal antibody - M_r apparent molecular weight - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We thank Mr. D. Mills and Mr. P. Stanley for help with the EM immunogold techniques. This work was supported by an Agricultural and Food Research Council grant and a studentship from the Science and Engineering Research Council.     

The host range and selectivity of a parasitic plant: Rhinanthus minor L.

Summary Rhinanthus minor (Yellow-rattle) is a widespread hemiparasitic plant of grassland habitats throughout Britain. Association analysis of the dune vegetation at Holme-next-the-Sea in eastern England revealed only two potential host plants through positive association. In contrast direct examination of the root systems revealed haustorial connections with 20 host species. The number of species parasitized by one plant ranged from one to seven. Data from another four sites in Britain and one in central Europe indicate that the natural host range of R. minor encompasses at least 50 species from 18 families with 22% in the Leguminosae and 30% in the Gramineae. Comparison of the number of haustorial connections made to each species with the abundance of roots in the soil shows that R. minor is a highly selective parasite, but that the selectivity is not consistent between populations or between plants from different parts of the same population. The reasons for host selectivity are discussed.     

Electron microscope autoradiography of14C photosynthate distribution at the haustorium-host interface in powdery mildew ofPisum sativum

Summary Haustorial complexes were isolated from leaves ofPisum sativum infected withErysiphe pisi and exposed to¹⁴CO₂ for 2 hours. The constituents of the isolated fraction were quantified and ultrastructurally described and the distribution of¹⁴C studied by electron microscope autoradiography and statistical treatment. Most (86%) of the isotope in the fraction was associated with haustorial complexes. Three classes of haustorial complexes were distinguished by degree of labelling and ultrastructure. Most of the haustorial complexes were termed healthy (i.e., they showed a normal ultrastructure) and were heavily labelled; necrotic complexes were unlabelled; and a class with intermediate labelling, modified extrahaustorial membrane and usually a normal haustorial cytoplasm was termed pre-necrotic. In healthy haustorial complexes the haustorial lobes and extrahaustorial membrane showed the highest grain densities and the body and extrahaustorial matrix were also significantly labelled. Comparison of the results suggest that ultrastructural modifications leading to necrosis were caused by dehydration which in turn determined reduction in photosynthate transfer. Other factors influencing transport into haustoria and the status of the extrahaustorial matrix are also discussed.U.V. fluorescence microscopy with acetamido-isothiocyanatostilbene salt was used to distinguish healthy and pre-necrotic haustorial complexes. It is recommended as a simple technique to monitor the functional quality of isolated haustorial complexes.     

Evolution of the secondary haustoria to a primary haustorium in the parasiticScrophulariaceae/Orobanchaceae

In the parasiticScrophulariaceae andOrobanchaceae, two types of contact organs exist: secondary and primary haustoria. Secondary haustoria are lateral organs, developing in large numbers and only when the seedling is fully established. In contrast, a primary haustorium represents the first developmental stage of the seedling itself. In the root system of the parasiticLesquereuxia syriaca (=Siphonostegia syriaca) there are only secondary haustoria, but a few of them apparently develop in a terminal position. This is achieved by transferring the haustorial initiation region closer to the root apex. One can interpret this as a transformation of the apical meristem into a meristematic haustorial tissue. On the condition that an extreme shortening (abbrevation) of the primary root could happen, we discuss the transformation of the terminal secondary into a primary haustorium.     

Differentiation-related proteins of the broad bean rust fungus Uromyces viciae-fabae,as revealed by high resolution two-dimensional polyacrylamide gel electrophoresis

On artificial polyethylene membranes providing a thigmotropic signal, uredospores of the broad bean rust fungus Uromyces viciae-fabae differentiated a series of infection structures which in nature are necessary to invade the host tissue through the stomata. Within 24 h germ tubes, appressoria, substomatal vesicles, infection hyphae and haustorial mother cells were developed successively. Alterations in protein metabolism during infection structure differentiation of this obligate plant pathogen were analyzed in the absence of the host plant by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver staining. The norm pattern representing the 2-DE protein patterns of the whole developmental sequence of infection structures of U. viciae-fabae showed 733 spots. During infection structure differentiation 55 proteins were newly formed, altered in quantity, or disappeared. Major alterations in the protein pattern occurred during uredospore germination and when infection hyphae were formed. Uredospore germination was characterized by a decrease of acidic proteins and an increase mainly of proteins with isoelectric points ranging from weakly acidic to basic.Abbreviations 2-DE two-dimensional polyacrylamide gel electrophoresis - DAPI 4,6-diamino-phenylindol - kDa kilo Dalton - pl isoelectric point - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Morphology,biology, and systematic relationships ofDesmaria (Loranthaceae)

Desmaria mutabilis is unique inLoranthaceae in having dimorphic shoots, the short shoots producing a terminal inflorescence. Other unusual features in the family are well differentiated bud scales and deciduousness. The normal position of mature plants on the trunks of large trees is shown to be a consequence of profuse vegetative reproduction from the epicortical roots, the predominant growth direction of the latter towards the trunk from the original site of establishment on a lateral branch, and the ability of epicortical roots to generate haustorial contacts through heavy host bark. The seedling is heterocotylar, one cotyledon being phanerocotylar, the other cryptocotylar and functioning as a haustorial organ in the endosperm. It is suggested thatDesmaria is a member of the primitive complex of loranthaceous genera which includesGaiadendron.     

Comparative ultrastructure of aecial and telial infections of the autoecious rust fungus Puccinia tuyutensis

This study demonstrates morphological differences between aecial and telial stages of the autoecious rust Puccinia tuyutensis. The aeciospores possess verrucose ornamentation while the teliospores have smooth surfaces. The aecial and telial haustoria of this rust produced in the mesophyll of Cressa cretica differ morphologically in the following respects:(1) the haustorial mother cell of telial haustorium is more differentiated than that of aecial haustorium and its wall at the penetration site is composed of 4 layers; (2) the aecial haustorium is filamentous in appearance and slightly constricted at the point of entry into the host cell, while the telial haustorium is clavate and possesses a narrow neck with a densely staining neckband and swollen body; (3) the neck of the telial haustorium is always associated with numerous vesicles while that of the aecial haustorium is not. Vascular tissue of host leaves is heavily invaded by aecial haustoria but not by telial haustoria.     

Direct cytoplasm-cytoplasm connection: an unusual host-parasite interaction of the tremelloid mycoparasiteTetragoniomyces uliginosus

Summary The cellular interaction ofTetragoniomyces uliginosus andRhizoctonia sp. was restudied by transmission electron microscopy. During the first stages of interaction a body of medium electron density is visible at the center of the haustorial apex in close association with the plasmalemma. A single micropore is produced between the haustorial filament and the host cell. Cytoplasmic connection via the pore always occurred. The pore membrane is continuous with the plasmalemma of both cells. The protoplasts of both the haustorium and the host cell fuse via the micropore. An electron transparent to dense body occlude the pore. Among basidiomycetes, direct connection between the parasite and host protoplasts represents a hitherto unknown type of parasitic interaction.Part 65 of the series Studies in Heterobasidiomycetes.     

Infection characteristics of a trematode in an estuarine isopod: influence of substratum

The estuarine isopod Cyathura carinata is a second intermediate host to microphallid trematodes, which use mud snails Hydrobia spp. and shorebirds as respectively first intermediate and final hosts. To identify processes responsible for infection patterns observed in C. carinata, a short-term microcosm experiment was conducted with both macroinvertebrates and one of their common parasites – Maritrema subdolum. Fine sand collected from two different shallow water sites was used to test if sediment type could affect infection rates. After 7 days at 25 °C, C. carinata from the substratum with the highest proportion of particles <125 m were more surface active and obtained significantly more M. subdolum individuals than isopods from the other sediment type. No parasite-induced effects on the hosts were found during this short-term experiment. The distribution pattern of microphallid cysts and mesocercariae inside the isopods revealed that M. subdolum cercariae primarily penetrated through the pleopods and afterwards located themselves in the middle-posterior region of the hosts body. Even if it was not possible to identify the factor responsible for the observed infection patterns (cercariae production and/or host behaviour), the results of this experiment indicate that small-scale factors, such as differences in substratum and associated features, may have considerable impact on infections of host populations.     

Components in the haustorial wall of the flax rust fungus,Melampsora lini, are labelled by three anti-calmodulin monoclonal antibodies

Summary Immunolocalisation studies, using flax leaf material infected with the flax rust fungus,Melampsora lini, and isolated haustorial complexes, have shown that three anti-calmodulin monoclonal antibodies bind to the haustorial wall of the fungus. The epitopes recognised by these antibodies are inserted into the wall during the early stages of haustorium development and remain in the wall throughout the life of the haustorium. The epitopes are present in both compatible and incompatible reactions and are oligosaccharide in nature. The results provide evidence for molecular differentiation within the haustorial complex ofM. lini.Abbreviations BMM butyl-methylmethacrylate - CaM calmodulin - FITC fluorescein isothiocyanate - MAb monoclonal antibody     

Self-penetration by the mycoparasiteDimargaris Cristalligena

Summary Ultrastructural evidence is presented for self-penetration by hyphae of the mycoparasiteDimargaris cristalligena. Plasmalemmal invagination occurs in a manner similar to that observed during penetration of a conventional host, and is accompanied by the synthesis of a penetration jacket. During self-penetration, however, there is little collar formation and the invading hypha fails to differentiate into a haustorial body.     

Water relations of the parasite: host relationship between the mistletoe Amyema linophyllum (Fenzl) Tieghem and Casuarina obesa Miq

Summary Seasonal and diurnal gas exchange and water relations of Amyema linophyllum and its host Casuarina obesa were studied at Gingin Western Australia. As recorded elsewhere for other species of mistletoe, stomatal conductances and transpiration rates were consistently higher in parasite than host, but assimilation rates did not differ significantly between partners, and water use efficiency was accordingly substantially lower in the parasite. Parallel responses of the species to environmental conditions suggested closely coordinated stomatal behaviour. However, sunlit and artifically shaded clumps of Amyema maintained high leaf conductances even when foliage fell below turgor loss point, yet their tissue capacitance values indicated maintenance of greater tissue water reserves during stress than in the host. Pressure-volume relationships indicated that differences in tissue water relations were unlikely to contribute significantly to the observed gradient in leaf water potential between partners. An experiment measuring changes in water potential of freshly detached host: parasite systems cut with the host shoot end immersed in water indicated that the haustorial junction was the principal site of resistance to transpiration-driven water flow into the parasite. A parallel experiment on intact attached shoots with mistletoe clumps enclosed and darkened just before dawn, demonstrated that, once the host commenced rapid transpiration, the water potential gradient between partners became reversed.     

Dynamic reorganization of microtubules and microfilaments in flax cells during the resistance response to flax rust infection

The cytoskeleton in plant cells is a dynamic structure that can rapidly respond to extracellular stimuli. Alteration of the organization of microtubules and actin microfilaments was examined in mesophyll cells of flax, Linum usitatissimum L., during attempted infection by the flax rust fungus, Melampsora lini (Ehrenb.) Lev. Flax leaves that had been inoculated with either a compatible (yielding a susceptible reaction) or an incompatible (yielding a resistant reaction) strain of M. lini were embedded in butyl-methylmethacrylate resin; sections of this material were immunofluorescently labelled with anti-tubulin or anti-actin and examined using confocal laser scanning microscopy. In uninfected leaves, microtubules in the mesophyll cells formed a transverse array in the cell cortex. Microfilaments radiated through the cytoplasm from the nucleus. In an incompatible interaction, microtubules and microfilaments were extensively reorganized in mesophyll cells that were in contact with fungal infection hyphae or haustorial mother cells before penetration of the cell by the infection peg. After the initiation of haustorium development, microtubules disappeared from the infected cells, and growth of the haustoria ceased. In an incompatible interaction, hypersensitive cell death occurred in more than 70% of infected cells but occurred in less than 20% of cells in compatible interactions. After the infected cell had undergone hypersensitive cell death, the cytoskeleton in neighbouring cells became focused on the walls shared with the necrotic cell. In compatible interactions, reorganization of the cytoskeleton was either not observed at all or was observed much less frequently up to 48 h after inoculation.Abbreviations FITC fluorescein isothiocyanate - WGA wheatgerm agglutinin We thank Dr. G.J. Lawrence for providing valuable discussions and materials.     

Light and Electron Microscopy of the Compatible Interaction Between Arabidopsis and the Downy Mildew Pathogen Peronospora parasitica

In this study, we focused on compatible interactions between Peronospora parasitica isolate Emoy‐2 and wild‐type (Oy‐0) and mutant (Ws‐eds1) Arabidopsis thaliana accessions by using light and transmission electron microscopy (TEM). Light microscopy of compatible interactions revealed that conidia germinated and penetrated through the anticlinal cell walls of two epidermal cells. Rapid spreading of the hyphal growth with formation of numerous haustoria within the mesophyll cells was subsequently followed by profuse sporulation in the absence of host cell necrosis on both wild‐type and mutant accessions. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were lobed with the diameter of 6–7 μm. Each haustorium was connected to intercellular hyphae in the absence of apparent haustorial neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. Callose‐like deposits were frequently observed at sites of penetration around the proximal region of the haustorial neck. Apart from a few callose ensheatments, no obvious response was observed in host cells following formation of haustoria. Most of mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cell of both accessions suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotroph oomycete differs considerably from responses to other pathogens such as necrotrophs.     

The interplay between host toxins and parasitism by Amoebophrya

The parasitic dinoflagellate Amoebophrya sp. ex Karlodinium veneficum was used to test two hypotheses: (1) infection of cells decreases with increasing host toxicity and (2) parasitism causes the catabolism of host toxin. To test the first hypothesis, host strains differing in toxin content were inoculated with dinospores of Amoebophrya sp. derived from infected cultures of toxic and non-toxic K. veneficum, with resulting infections assessed following 24-h incubations. Contrary to expectations, infection of K. veneficum by Amoebophrya sp. was positively correlated with host toxicity. To examine the second hypothesis, synchronous infection with >80% of cells being parasitized was induced using a toxic strain of K. veneficum, and total toxin concentration (intracellular plus extracellular levels of KmTX1) was followed over the 3-day infection cycle. Toxin content ml⁻¹ increased with growth of K. veneficum in uninfected control cultures, but declined in infected cultures as the parasite completed its life cycle. On a cellular basis, toxin content of infected and uninfected cultures differed little during the experiment, suggesting that the parasite does not actively catabolise host toxin. Rather, infection appears to promote degradation of toxins via death of host cells and subsequent bacterial activity. Results indicate that Amoebophrya sp. ex K. veneficum has greater potential to impact toxic strains relative to non-toxic host strains in natural systems. Thus, Amoebophrya sp. ex. K. veneficum may limit the occurrence of toxic K. veneficum blooms in marine and estuarine environments, while simultaneously functioning as a pathway for dissipation of host toxin.     

Nodulation of specific legumes is controlled by several distinct loci in Rhizobium trifolii

Summary Three distinct loci (designated regions III, IV and V) were identified in the 14 kb Nod region of Rhizobium trifolii strain ANU843 and were found to determine the host range characteristics of this strain. Deletion of region III or region V only from the 14 kb Nod region affected clover nodulation capacity. The introduction to R. Leguminosarum of DNA fragments on multicopy vectors carrying regions III, IV and V (but not smaller fragments) extended the host range of R. leguminosarum so that infection threads and nodules occurred on white clover plants. The same DNA fragments were introduced to the Sym plasmid-cured strain (ANU845) carrying the R. meliloti recombinant nodulation plasmid pRmSL26. Plasmid pRmSL26 alone does not confer root hair curling or nodulation on clover plants. However, the introduction to ANU845 (pRmSL26) of a 1.4 kb fragment carrying R. trifolii region IV only, resulted in the phenotypic activation of marked root hair curling ability to this strain on clovers but no infection events or nodules resulted. Only the transfer of regions III, IV and V to strain ANU845 (pRmSL26) conferred normal nodulation and host range ability of the original wild type R. trifolii strain. These results indicate that the host range genes determine the outcome of early plant-bacterial interactions primarily at the stage of root hair curling and infection.     

马铃薯对晚疫病水平抗性的组织细胞学观察

以马铃薯晚疫病水平抗性品种LBr-12和感病品种费乌瑞它为材料,采用普通光学和电子显微镜技术,系统研究了马铃薯与晚疫病菌(致病疫霉)互作的组织细胞学反应特征。观察结果显示:(1)接种后,水平抗性材料LBr-12出现过敏反应,病菌被限制在侵染点的几个细胞中,菌丝产生较少的分支和吸器。(2)感病品种费乌瑞它被侵染细胞呈蔓延趋势,菌丝产生较多的分支和吸器。(3)电镜观察发现,抗病品种上病菌的胞间菌丝、吸器母细胞、吸器在细胞和亚细胞水平均发生了一系列异常变化,包括原生质的电子致密度加深、液泡增多变大、菌丝细胞壁不规则增厚、细胞器排列紊乱及解体、吸器母细胞及吸器形态异常、病菌最终畸形坏死,同时发现抗病品种受病菌侵染时可迅速产生结构防卫反应,形成的细胞壁沉积物使胞壁极度增厚或细胞膜上产生乳突状结构。