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.     

条锈菌与慢锈小麦品种互作的超微结构*

条锈菌与慢锈小麦品种互作的超微结构研究表明,随着慢锈性的表达,条锈菌的胞间菌丝发育受抑,细胞器泡囊化解体。吸器母细胞和吸器发育受阻,最终解体、坏死。吸器的受抑、坏死主要表现在吸器体形成后期。在锈菌与寄主互作的交界面,吸器外质膜皱褶、电子致密度增高,吸器外间质加宽,并有大量电子致密度加深的物质沉积。与抗病品种相比, 慢锈品种中病菌受抑、坏死程度轻,寄主细胞的过敏性坏死机率也较低。     

Observations on the ultrastructure of the uredinial stage ofPuccinia polypogonis onPolypogon monspeliensis

The ultrastructure of the uredinial stage of the rust fungus,Puccinia polypogonis onPolypogon monspeliensis is described, using scanning and transmission electron microscopy. This study examined the urediniospores, intercellular hyphae, and haustoria of the fungus. The formation and structure of urediniospores is similar to those of otherPuccinia species. The ultrastructure of intercellular hyphae and haustoria is similar to those of other rust fungi, but with some differences. No modifications are observed in the wall of the haustorial mother cells during penetration. A collar is found only around old haustoria. In most cases, one nucleus is detected inside the haustorial body and no nucleoli are seen in the nuclei of intercellular hyphae and haustoria. The host-parasite interface, including extrahaustorial matrix and extrahaustorial membrane, is also discussed and compared with those of other rust fungi.     

Identification of a protein from rust fungi transferred from haustoria into infected plant cells

The formation of haustoria is one of the hallmarks of the interaction of obligate biotrophic fungi with their host plants. In addition to their role in nutrient uptake, it is hypothesized that haustoria are actively involved in establishing and maintaining the biotrophic relationship. We have identified a 24.3-kDa protein that exhibited a very unusual allocation. Rust transferred protein 1 from Uromyces fabae (Uf-RTP1p) was not only detected in the host parasite interface, the extrahaustorial matrix, but also inside infected plant cells by immunofluorescence and electron microscopy. Uf-RTP1p does not exhibit any similarity to sequences currently listed in the public databases. However, we identified a homolog of Uf-RTP1p in the related rust fungus Uromyces striatus (Us-RTP1p). The localization of Uf-RTP1p and Us-RTP1p inside infected plant cells was confirmed, using four independently raised polyclonal antibodies. Depending on the developmental stage of haustoria, Uf-RTP1p was found in increasing amounts in host cells, including the host nucleus. Putative nuclear localization signals (NLS) were found in the predicted RTP1p sequences. However, functional efficiency could only be verified for the Uf-RTP1p NLS by means of green fluorescent protein fusions in transformed tobacco protoplasts. Western blot analysis indicated that Uf-RTP1p and Us-RTP1p most likely enter the host cell as N-glycosylated proteins. However, the mechanism by which they cross the extrahaustorial membrane and accumulate in the host cytoplasm is unknown. The localization of RTP1p suggests that it might play an important role in the maintenance of the biotrophic interaction.     

Isolation by ConA binding of haustoria from different rust fungi and comparison of their surface qualities

Summary Rust haustoria isolated from infected leaf tissue strongly bind to ConA. This property was exploited to purify them by affinity chromatography on a ConA-Sepharose macrobead column. Haustoria were obtained with more than 90% purity and yields of up to 50%. Binding of haustoria to the column was partially inhibited by a ConA-specific sugar, methyl -D-mannopyranoside. Compared to ConA,Lens culinaris agglutinin and wheat germ agglutinin were less efficient affinity ligands. Using ConA-Sepharose, rust haustoria from a variety of sources could be isolated with equal efficiency, indicating that they have similar carbohydrate surface properties. The haustoria maintained their typical shape after the isolation procedure, which suggests a rather rigid wall structure. The morphology of haustoria was characteristic both for a given species and the nuclear condition of the rust mycelium. Electron microscopy of isolated haustoria revealed an intact haustorial wall surrounded by a fibrillar layer presumably derived from the extrahaustorial matrix. The matrix thus appears to represent a layer with gel-like properties which is rich in ConA-binding carbohydrates and connected to the haustorial wall but not to the host-derived extrahaustorial membrane.Abbreviations ConA Concanavalin A - LCA Lens culinaris agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - DAPI 4,6-diamidinophenylindol×2 HCl     

Immunogold localization of THRGP-like epitopes in the haustorial interface of obligate,biotrophic fungi on monocots

Summary Immunoelectron microscopy was used to determine the subcellular distribution of threonine-hydroxyproline-rich glycoprotein (THRGP) epitopes in host-parasite interactions between obligate, biotrophic fungi and cereals. Infection sites of stem rust (Puccinia graminis f. sp.tritici) and leaf rust (Puccinia recondita) on primary leaves of wheat (Triticum aestivum), as well as of powdery mildew (Erysiphe graminis f. sp.hordei) on coleoptiles of barley (Hordeum vulgare), wete probed with a polyclonal antiserum to maize THRGP. A few immunogold particles were found over the cell walls of wheat mesophyll tissue and barley coleoptile epidermis. Unlike previous examples in dicot plants, no enhanced accumulation of THRGP was observed in cereal cell walls adjacent to sites of pathogen ingress. Instead, the most pronounced accumulation of THRGP-like molecules occurred over the extrahaustorial matrix in both incompatible and compatible plant-pathogen interactions. For powdery mildew of barley, immunogold staining was distinctly increased over the center of the penetration sites; however, no labeling was found over papillae that formed during incompatible and compatible interactions. In addition, no cross-reactivity of the anti-THRGP antiserum with intercellularly growing rust pathogens was observed. The highly localized deposition of THRGP-like molecules in the extrahaustorial matrix suggests that the host plant establishes a modified barrier between itself and the pathogen.Abbreviations C chloroplast - EC plant epidermal cell - EM extrahaustorial membrane - EMA extrahaustorial matrix - GO Golgi body - GRP glycine-rich protein - HP high pressure - HRGP hydroxyprolinerich glycoprotein - Hyp hydroxyproline - LT low temperature - PBS phosphate-buffered saline - PBST PBS with Tween-20 - THRGP threonine-hydroxyproline-rich glycoprotein - VA vesicular arbuscular     

Sequential deposition of plant glycoproteins and polysaccharides at the host-parasite interface ofUromyces vignae andVigna sinensis

Summary Monokaryotic haustoria (M-haustoria) ofUromyces vignae inVigna sinensis cells are surrounded by an extrahaustorial matrix (ema) and the invaginated host plasmalemma, the extrahaustorial membrane (ehrn). The ema was characterized with antibodies against components of the plant cell wall; the ema contained hydroxyproline-rich glycoproteins and arabinogalactans/arabinogalactan proteins, both at a higher concentration close to the ehm. Haustoria with large vacuoles had the ema encased by additional layers. An electron-translucent inner layer deposited on top of the ema contained arabinogalactans/arabinogalactan proteins, hydroxyproline-rich glycoproteins, and callose. The inner layer was surrounded by an electron-translucent middle layer with numerous dark inclusions, rich in pectin and fucose bound to xyloglucans. Finally, a more electron-dense outer layer containing arabinogalactans/arabinogalactan proteins and hydroxyproline-rich glycoproteins encased the whole structure. These polysaccharides, with the exception of callose and un-esterified pectin, were also found in the plant Golgi apparatus. The polysaccharides were synthesized in the trans Golgi cisternae and secreted into the host-parasite interface. The secretory events seem to be coupled to endocytosis since numerous coated pits were found on the ehm too. The pits were elongated, sometimes formed tubules and the coat reacted with an antibody against plant clathrin. Our results suggest intensive membrane recycling around haustoria, together with the secretion of cell wall material, which in the case of more or less vacuolated haustoria seems to be responsible for encasementAbbreviations AG/AGP arabinogalactans and arabinogalactan proteins - BSA bovine serum albumin - ehm extrahaustorial membrane - ema extrahaustorial matrix - HRGP_2b hydroxyproline rich glycoproteins - M-haustorium monokaryotic haustorium - TBS tris buffered saline     

Heat sensitivity of mycelium and haustoria in bean leaves infected with bean rust

Bean leaves experimentally inoculated with bean rust were subjected to heat treatment at 50° for 20 s, at different times after inoculation, and the modifications induced by heating in the pathogen and host cells were studied by electron microscopy, at different times after the treatment. This induced no alterations either in healthy or infected leaf cells. On the contrary, it caused severe ultrastructural alterations in the pathogen. These alterations were already visible in the haustoria and hyphae at the end of the treatment, became progressively more severe with time, and led to the death of the pathogen two days after the treatment. From this time onwards the haustoria appeared shrunken, extremely electron-dense, and encased in a mass of granular material, seemingly secreted by the host cell. No encasement was ever found around haustoria apparently dead by senescence in untreated leaves.     

Development and Structure of the Haustorium of the Parasite Rhamphicarpa fistulosa (Scrophulariaceae)

Rhamphicarpa fistulosa (Hochst.) Benth. (Scrophulariaceae), a parasite of African cereals, develops secondary haustoria which penetrate the roots of the host plant. Light and electron microscopy have been used to study the structure and development of haustoria in this species, which, until now, have not been well characterized. Haustoria are initiated in the hypodermis of the parasite roots. A meristematic strand is developed between the parasite root stele and the host-parasite interface. From this strand, cells differentiate into xylem elements after penetration of the host root. Xylem differentiation follows an acropetal pattern. Mature haustoria are characterized by a continuous xylem bridge between water conducting elements of parasite and host. A detailed study of the hostparasite interface revealed the presence of collapsed and compressed host cells at the lateral interface (between parasite cells and host cortex), whereas the central interface between parasite cells and the host stele is almost devoid of host cell remnants. Implications of these observations for the penetration mechanisms are discussed.     

Elektronenmikroskopische Untersuchungen des Gerstenmehltaus (Erysiphe graminis DC f. sp. hordei Marchal) nach Resistenzinduktion mit mikrobiellen Stoffwechselprodukten

Electron microscopical studies on Mildew of Barley (Erysiphe graminis DC f. sp. hordei Marchal) after induced Resistance with Products of Microbial Metabolism Ultrastructural studies of the infection progress of barley by Erysiphe graminis after induction of resistance with products of microbial metabolism showed that pathogen development was affected through inducer activated defence mechanisms. The formation of papillae-like structures and an accumulation of electron dense material below the host cell wall at the penetration site appeared to be associated with reduced penetration. The haustoria were also altered in the extrahaustorial membrane and electron dense material accumulated at the cell wall of haustorial body and neck, thus apparently impairing efficient functioning and allowing only limited growth of the pathogen.     

Biogenesis of a specialized plant-fungal interface during host cell internalization of Golovinomyces orontii haustoria

Powdery mildew fungi are biotrophic pathogens that require living plant cells for their growth and reproduction. Elaboration of a specialized cell called a haustorium is essential for their pathogenesis, providing a portal into host cells for nutrient uptake and delivery of virulence effectors. Haustoria are enveloped by a modified plant plasma membrane, the extrahaustorial membrane (EHM), and an extrahaustorial matrix (EHMx), across which molecular exchange must occur, but the origin and composition of this interfacial zone remains obscure. Here we present a method for isolating Golovinomyces orontii haustoria from Arabidopsis leaves and an ultrastructural characterization of the haustorial interface. Haustoria were progressively encased by deposits of plant cell wall polymers, delivered by secretory vesicles and multivesicular bodies (MVBs) that ultimately become entrapped within the encasement. The EHM and EHMx were not labelled by antibodies recognizing eight plant cell wall and plasma membrane antigens. However, plant resistance protein RPW8.2 was specifically recruited to the EHMs of mature haustoria. Fungal cell wall-associated molecular patterns such as chitin and β-1,3-glucans were exposed at the surface of haustoria. Fungal MVBs were abundant in haustoria and putative exosome vesicles were detected in the paramural space and EHMx, suggesting the existence of an exosome-mediated secretion pathway.     

植物专性寄生菌吸器功能研究现状北大核心CSCD

吸器是专性寄生真菌和卵菌的菌丝产生的一种短小分支变态结构,由吸器体、吸器外间质和吸器外质膜3部分组成。吸器不仅仅是吸收和转运寄主植物的营养物质的功能,它在病原菌生物合成、抑制寄主的防御反应等方面也具有不同程度的作用。对吸器的深入了解将有助于更好地认识、控制专性寄生菌。本文综述了吸器关于营养吸收与致病性方面的功能,讨论了有待解决的问题及今后的研究趋势。     

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.     

Attachment of the parasitic weed dodder to the host

Summary.  The parasitic weed dodder (Cuscuta pentagona L.) invades a number of potential host species, but the mechanisms responsible for ensuring tight adhesion to the wide variety of host surfaces have yet to be identified. In this study, a battery of microscopy protocols is used to examine the host–parasite interface in an effort to deduce these mechanisms. As the dodder shoot approaches the host tissue, epidermal cells in the parasite shoot elongate and differentiate into secretory type trichomes. The trichome cell walls are malleable, allowing them to elongate towards the host and bend their walls to conform to the shape of the host cell surface. The presence of osmiophilic particles (probable cell-wall-loosening complexes) at far greater numbers than found in other species presages the expansion and malleable nature of the epidermal cells. In addition to the changes in cell shape, the dodder trichome cells secrete an electron-opaque cementing substance that covers the host–parasite interface. When probed with antibodies that recognize cell wall components, the cement reacted only with antibodies that recognize chiefly de-esterified pectins but not other common wall constituents. These data indicate that dodder utilizes both a cementing layer of pectin and a radically modified epidermal cell wall to secure the parasite to the perspective host. Received January 29, 2001 Accepted November 28, 2001     

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     

Abnormal callose response phenotype and hypersusceptibility to Peronospoara parasitica in defence-compromised arabidopsis nim1-1 and salicylate hydroxylase-expressing plants

To investigate the impact of induced host defenses on the virulence of a compatible Peronospora parasitica strain on Arabidopsis thaliana, we examined growth and development of this pathogen in nim1-1 mutants and transgenic salicylate hydroxylase plants. These plants are unable to respond to or accumulate salicylic acid (SA), respectively, are defective in expression of systemic acquired resistance (SAR), and permit partial growth of some normally avirulent pathogens. We dissected the P. parasitica life cycle into nine stages and compared its progression through these stages in the defense-compromised hosts and in wild-type plants. NahG plants supported the greatest accumulation of pathogen biomass and conidiophore production, followed by nim1-1 and then wild-type plants. Unlike the wild type, NahG and nim1-1 plants showed little induction of the SAR gene PR-1 after colonization with P parasitica, which is similar to our previous observations. We examined the frequency and morphology of callose deposits around parasite haustoria and found significant differences between the three hosts. NahG plants showed a lower fraction of haustoria surrounded by thick callose encasements and a much higher fraction of haustoria with callose limited to thin collars around haustorial necks compared to wild type, whereas nim1-1 plants were intermediate between NahG and wild type. Chemical induction of SAR in plants colonized by P. parasitica converted the extrahaustorial callose phenotype in NahG to resemble closely the wild-type pattern, but had no effect on nim1-1 plants. These results suggest that extrahaustorial callose deposition is influenced by the presence or lack of SA and that this response may be sensitive to the NIM1/NPR1 pathway. Additionally, the enhanced susceptibility displayed by nim1-1 and NahG plants shows that even wild-type susceptible hosts exert defense functions that reduce disease severity and pathogen fitness.     

Characterization of the membrane-associated HaRxL17 Hpa effector candidate

We examined changes to subcellular architecture during the compatible interaction between the biotroph pathogen Hyaloperonospora arabidopsidis (Hpa) and its host Arabidopsis. Live-cell imaging highlighted rearrangements in plant cell membranes upon infection. In particular, the tonoplast appeared close to the extrahaustorial membrane surrounding the haustorium. We investigated the subcellular localization patterns of Hpa RxLR effector candidates (HaRxLs) in planta. This subcellular localization screening led to the identification of an extrahaustorial membrane-localized effector, HaRxL17 that when stably expressed in Arabidopsis increased plant susceptibility to Hpa during compatible and incompatible interactions. Here, we report that the N-terminal part of HaRxL17 is sufficient to target the plant cell membranes. We showed that both C- or N-terminal fluorescent-tagged HaRxL17 localizes around Hpa haustoria, in early and in late stages of infection. As with Hpa infection, GFP-HaRxL17 also localizes around haustoria during infection with Albugo laibachii. Thus, HaRxL17 that increases plant susceptibility to Hpa during both compatible and incompatible interactions, localizes around oomycete haustoria when stably expressed in Arabidopsis.